Heart-lung Block Research Articles

Hyperacute experimental model of rat lung transplantation using a coronary shunt cannula.

Lung transplantation is a well-established treatment of end-stage lung disease. A rodent model is an inexpensive way to collect biological data from a living model after lung transplantation. However, mastering the surgical technique takes time owing to the small organ size. To conduct rat lung transplantation using a shunt cannula (SC) or modified cannula (MC) and assess their efficacy. Rat lung transplantation was performed in 11 animals in the SC group and 12 in the MC group. We devised a method of rat lung transplantation using a coronary SC for coronary artery bypass surgery as an anastomosis of pulmonary arteriovenous vessels and bronchioles. The same surgeon performed all surgical procedures in the donor and recipient rats without using a magnifying glass. The success rate of lung transplantation, operating time, and PaO2 values were compared after 2-h reperfusion after transplantation. Ten and 12 lungs were successfully transplanted in the SC and MC groups, respectively. In the SC group, one animal had cardiac arrest within 1 h after reperfusion owing to bleeding during pulmonary vein anastomosis. The operating time for the removal of the heart-lung block from the donor and preparation of the left lung graft was 26.8 ± 2.3 and 25.7 ± 1.3 min in the SC and MC groups, respectively (P = 0.21). The time required for left lung transplantation in the recipients was 37.5 ± 2.8 min and 35.9 ± 1.4 min in the SC and MC groups, respectively (P = 0.12). PaO2 values at 2 h after reperfusion were 456.2 ± 25.5 and 461.2 ± 21.5 mmHg in the SC and MC groups, respectively (P = 0.63), without difference between the groups. A hyperacute rat lung transplantation model using a coronary SC was created using a simple technique. The MC was inexpensive, easy to prepare, and simple to operate.

Male versus female inflammatory response after brain death model followed by ex vivo lung perfusion

BackgroundEx vivo lung perfusion (EVLP) is a useful tool for assessing lung grafts quality before transplantation. Studies indicate that donor sex is as an important factor for transplant outcome, as females present higher inflammatory response to brain death (BD) than males. Here, we investigated sex differences in the lungs of rats subjected to BD followed by EVLP.MethodsMale and female Wistar rats were subjected to BD, and as controls sham animals. Arterial blood was sampled for gas analysis. Heart–lung blocks were kept in cold storage (1 h) and normothermic EVLP carried out (4 h), meanwhile ventilation parameters were recorded. Perfusate was sampled for gas analysis and IL-1β levels. Leukocyte infiltration, myeloperoxidase presence, IL-1β gene expression, and long-term release in lung culture (explant) were evaluated.ResultsBrain dead females presented a low lung function after BD, compared to BD-males; however, at the end of the EVLP period oxygenation capacity decreased in all BD groups. Overall, ventilation parameters were maintained in all groups. After EVLP lung infiltrate was higher in brain dead females, with higher neutrophil content, and accompanied by high IL-1β levels, with increased gene expression and concentration in the culture medium (explant) 24 h after EVLP. Female rats presented higher lung inflammation after BD than male rats. Despite maintaining lung function and ventilation mechanics parameters for 4 h, EVLP was not able to alter this profile.ConclusionIn this context, further studies should focus on therapeutic measures to control inflammation in donor or during EVLP to increase lung quality.

Novel Polymerized Human Serum Albumin For Ex Vivo Lung Perfusion.

Ex vivo lung perfusion (EVLP) is a method of organ preservation to expand the donor pool by allowing organ assessment and repair. Perfusion solution composition is crucial to maintaining and improving organ function during EVLP. EVLP compared perfusates supplemented with either polymeric human serum albumin (PolyHSA) or standard human serum albumin (HSA). Rat heart-lung blocks underwent normothermic EVLP (37°C) for 120 minutes using perfusate with 4% HSA or 4% PolyHSA synthesized at a 50:1 or 60:1 molar ratio of glutaraldehyde to PolyHSA. Oxygen delivery, lung compliance, pulmonary vascular resistance (PVR), wet-to-dry ratio, and lung weight were measured. Perfusion solution type (HSA or PolyHSA) significantly impacted end-organ metrics. Oxygen delivery, lung compliance, and PVR were comparable among groups ( P > 0.05). Wet-to-dry ratio increased in the HSA group compared to the PolyHSA groups (both P < 0.05) suggesting edema formation. Wet-to-dry ratio was most favorable in the 60:1 PolyHSA-treated lungs compared to HSA ( P < 0.05). Compared to using HSA, PolyHSA significantly lessened lung edema. Our data confirm that the physical properties of perfusate plasma substitutes significantly impact oncotic pressure and the development of tissue injury and edema. Our findings demonstrate the importance of perfusion solutions and PolyHSA is an excellent candidate macromolecule to limit pulmonary edema. http://links.lww.com/ASAIO/A980.

State of the art - Extracorporeal membrane oxygenation as a bridge to thoracic transplantation.

Extracorporeal membrane oxygenation (ECMO) has revolutionized the treatment of refractory cardiac and respiratory failure, and its use continues to increase, particularly in adults. However, ECMO-related morbidity and mortality remain high. In this review, we investigate and expand upon the current state of the art in thoracic transplant and extracorporeal life support (ELS). In particular, we examine recent increase in incidence of heart transplant in patients supported by ECMO; the potential changes in patient care and selection for transplant in the years prior to updated United Network for Organ Sharing (UNOS) organ allocation guidelines versus those in the years following, particularly where these guidelines pertain to ECMO; and the newly revived practice of heart-lung block transplants (HLT) and the prevalence and utility of ECMO support in patients listed for HLT. Our findings highlight encouraging outcomes in patients bridged to transplant with ECMO, considerable changes in treatment surrounding the updated UNOS guidelines, and complex, diverse outcomes among different centers in their care for increasingly ill patients listed for thoracic transplant.

Study of variations in the broncho-arterial pedicles of the upper right lung lobe

Bronchial distribution and functional arterial vascularization of the upper lobe of the right lung are subject to many anatomical variations. The control of these variations is essential for endoscopic and agiographic examinations. It also offers a better guarantee for safe and controlled surgery. In this preliminary work, the exploitation of 15 heart-lung blocks treated by the injection corrosion method allowed us to study the general arrangement of the broncho-arteries of the right upper lung lobe and their anatomical variations in the Senegalese population. Our results were as follows: the right upper lobar bronchus was born on average at 1.25 cm from the tracheal bifurcation, with an average length of 1.13 cm. It ended with trifurcation into apical (B1), dorsal (B2) and ventral (B3) segmental bronchi in 10 cases (66.66%); in 3 cases (20%), it ended with bifurcation into the dorsal segmental bronchus and the apico-ventral trunk (B1+B3) (1 case), the apico-dorsal trunk (B1+B2) and the ventral segmental bronchus (B3), finally, in ventral and dorsal segmentary bronchi giving each one an apical branch (1 case); in a last case, it ended by quadrifurcation, giving an external parabronche. The right upper lobe was vascularized by 1 to 4 arteries, with eight modes of vascularization. It received more frequently two arteries. The anterior mediastinal artery was the most common (100%). These results allowed us to discuss anatomical variations in the bronchial tree of the right upper lung lobe and the pulmonary arterial distribution in that lobe. These variations must be taken into account during endoscopic examinations of imaging and surgery of pulmonary excision, under penalty of accidents.

Cytosolic DNA Sensors as a New Pharmacological Target in EVLP

<h3>Purpose</h3> Innate immunity is a key component of the inflammatory responses ensuing ischemia/reperfusion (I/R) injury. Cell damage and death caused by I/R promotes the release of danger-associated molecular patterns (DAMPs) recognized by specific pattern recognition receptors (PRRs). DNA released from nucleus and mitochondria of injured cells is a known DAMP, which sustains inflammation and cell death when recognized by specific PRRs like cytosolic DNA (cDNA) sensors cGAS and AIM2. The cGAS/STING pathway induces the production of type I interferons (IFNs) and IFN stimulated genes (ISGs). The role of cDNA sensors in reperfused lung is currently not characterized. Thus, using the rat <i>ex vivo</i> lung perfusion (EVLP) and lung transplantation (LTx) models, we begin to investigate the role of cGAS/STING in the early inflammatory events of lung I/R. <h3>Methods</h3> Heart-Lung blocks from male Sprague-Dawley rats were cannulated in the pulmonary artery and left atrium and assigned to four groups (n=5/6 rats/group). Cold ischemia (CI): under mechanical ventilation, the lungs were flushed with 4°C Perfadex. The heart-lung block was stored inflated (FiO2 0.5) for 7h in 4°C Perfadex. Warm ischemia (WI): After cannulation, the lungs were deflated for 1h at room temperature, flushed and stored as above for 6h. CI+EVLP: lungs were harvested as above, stored at 4°C for 2h, followed by 3-hour EVLP, and stored again (2h) in 4°C Perfadex. WI+EVLP: after 1 hour WI, lungs were flushed and stored at 4°C for 1 hour, followed by 3h EVLP, and stored again (2h) in 4°C Perfadex. Right lungs were used as controls and left lungs were transplanted and sampled 2h after LTx. We analyzed by RT-PCR and western blot the expression of the cytosolic DNA sensors cGAS, STING and AIM2 and by RT-PCR the expression of the downstream gene <i>Ifnb</i>, and the ISGs <i>Ifit1</i> and <i>Isg15</i> in whole lung tissue<i>.</i> <h3>Results</h3> cGAS, STING and AIM2 proteins are expressed in lung after the ischemic period. Their mRNA expression is strongly induced by the reperfusion (EVLP, LTx or EVLP+LTx) regardless of the conditions of preservation (CI or WI). We observed a similar behavior for the mRNA expression of <i>Ifnb</i> and the ISGs <i>Ifit1</i> and <i>Isg15</i>. <h3>Conclusion</h3> These preliminary data provide evidence that cytosolic DNA sensors, especially the cGAS-STING axis, may play a prominent role in the early inflammation of the reperfused lung regardless of the preservation conditions.

Lung Function and Inflammatory Profiling of Damaged Rat Donor Lungs Following EVLP Heat Stress with Different Temperatures

<h3>Purpose</h3> Ex-vivo lung perfusion (EVLP) is a validated technique allowing evaluation and reconditioning of damaged donor lungs to make them suitable for lung transplantation. Moderate hyperthermia is known to exert cytoprotective effect, via induction and accumulation of specific proteins, known as heat shock proteins (HSPs). We hypothesized that thermal preconditioning (TP) with a well-calibrated heat stress applied during EVLP could improve lung graft by reduction of oxidative stress, cell death and inflammation. <h3>Methods</h3> Male rats were randomly assigned into 5 groups: controls (Ctrl, n=5) and four heat stress groups (HS of 41.5°C; 42.0°C; 42.5°C or 43.5°C, n=5). In all groups, lungs were kept <i>in situ</i> for 1h at room temperature, then flushed with cold Perfadex. Heart-lung block was harvested and kept at 4°C for 1h, then mounted on the EVLP system for 3h. In Ctrl group, the temperature perfusion solution was kept at 37°C the all-time. In the HS groups, a heat shock was done for 30 minutes after 1h of EVLP by heating the perfusion solution at different temperature. At the end of EVLP, lungs were snap-frozen and kept at -80°C for further tissue analysis. <h3>Results</h3> Static pulmonary compliance SPC ratio (180 min to 60 min of EVLP) was significantly increased in the 41.5°C group compared to Ctrl group, while it was significantly reduced in 43.5°C group. The weight gain was significantly reduced in 41.5°C, whereas increased in 43.5°C group. In all HS groups, except in 43.5°C, protein levels of inducible HSP72 and Heme oxygenase-1 (HO-1) were significantly increased compared to Ctrl. Reduction of caspase 3 activation (apoptosis marker) was seen in all groups but reached significant level only in 41.5°C and 43.5°C groups. In all HS groups, we could see a reduction of inflammatory mediators such as, IL-1α, IL-1β, and TNF-α, a reduction of the release of CC16 (a marker of lung epithelial injury), as well as a reduction of IL-1β in Steen solution. <h3>Conclusion</h3> Thermal preconditioning during EVLP can decrease apoptotic cell death and inflammation of damaged lungs. These findings demonstrate that EVLP in association with well calibrated thermal preconditioning (41.5°C) could be a promising therapeutic platform improving lung quality of damaged donor lungs.

Lung Function and Inflammatory Profiling of Damaged Rat Donor Lungs Following EVLP Thermal Preconditioning

<h3>Purpose</h3> Ex-vivo lung perfusion (EVLP) is a validated technique allowing evaluation and reconditioning of damaged donor lungs to make them suitable for transplantation. Moderate hyperthermia is known to exert cytoprotective effect, via induction and accumulation of specific proteins, known as heat shock proteins (HSPs). We hypothesized that thermal preconditioning (TP) with a well-calibrated heat stress applied during EVLP could improve lung graft by reduction of oxidative stress, cell death and inflammation. <h3>Methods</h3> Male rats were randomly assigned into 2 groups: controls (Ctrl, n=5) or TP (n=5). In both groups, lungs were kept <i>in situ</i> for 1h at room temperature, then flushed with cold Perfadex. Heart-lung block was harvested and kept at 4°C for 1h, then mounted on the EVLP system for 3h. In Ctrl group, the temperature perfusion solution was kept at 37°C the all-time. In the TP group, a heat shock was done for 30 minutes after 1h of EVLP by heating the perfusion solution at 41.5°C. At the end of EVLP, lungs were snap-frozen and kept at -80°C for further tissue analysis. <h3>Results</h3> Static pulmonary compliance, peak inspiratory pressure, and oxygenation capacity were similar in both groups. We were able to induce HSPs in the TP group (<i>Hspa1a</i> and <i>Hmox-1</i>). In the TP group, we observed a reduction of oxidative stress (carbonyls and MDA), a decrease of apoptosis (Caspase 3 and 7) and a decrease of pro-inflammatory NF-κB transcription factor as well as the expression of inflammation mediators such as TNF-α, IL-1α, IL-1β, CXCL-1 and CCL-2. <h3>Conclusion</h3> Thermal preconditioning during EVLP decreased oxidative stress, apoptotic cell death, and inflammation of damaged lungs. These findings demonstrate that EVLP in association with thermal preconditioning could be a promising therapeutic platform improving lung quality of damaged donor lungs.

Development and Evaluation of a Novel Combined Perfusion Decellularization Heart-Lung Model for Tissue Engineering of Bioartificial Heart-Lung Scaffolds

<h3>Purpose</h3> Tissue engineering provides a new concept for organ and tissue replacement, and may represent a new approach to generate native-like substitutes for extensive cardiothoracic disorders. Bioengineered transplantable heart-lung scaffolds could be potentially lifesaving in a large number of congenital and acquired cardiothoracic disorders including terminal heart-lung disease. The aim of this project was to evaluate mechanical and biochemical properties of the native heart and lung, to optimize a decellularization protocol to obtain a suitable heart-lung acellular grafts that can be re-seeded with cells and implanted without need of immunosuppression, and potentially to be able to create an artificial heart-lung scaffold made up by decellularization of native heart-lung blocks seeded with an appropriate source of mononuclear or stromal cells to accurately mimic the structure and function of a native heart-lung substitute. <h3>Methods</h3> We decellularized rat hearts (n = 5) and heart-lung organ-blocs (n = 5) by coronary and tracheal perfusion with ionic detergents in a modified Langendorff circuit. We confirmed decellularization by histology, transmission electron microscopy and fluorescence microscopy. <h3>Results</h3> In the present project we were able to achieve 100% decellularization of the heart and heart-lung organ-bloc <i>(Figure 1)</i>. Decellularized heart-lung organ-blocs lacked intracellular components but retained specific collagen fibers and mechanical integrity. <h3>Conclusion</h3> We present a novel model of a combined perfusion and decellularization of the heart and heart-lung organ-blocs. This model is a first step in the direction of creating bioengineered transplantable heart-lung scaffolds. We believe that further developing of this technology could provide a life-saving conduit, significantly reducing the risks of heart-lung failure surgery, and improve post-operative quality of life.

Right lung transplantation with a left-to-right inverted anastomosis in a rat model

ObjectiveRight lung transplantation in rats has been attempted occasionally, but the technical complexity makes it challenging to apply routinely. Additionally, basic research on inverted lobar lung transplantation is scarce because of the lack of a cost-effective experimental model. We first reported right lung transplantation in a rat model using left-to-right inverted anastomosis to imitate the principle of clinically inverted lung transplantation. MethodsRight lung transplantation was performed in 10 consecutive rats. By using a 3-cuff technique, the left lung of the donor rat was implanted into the right thoracic cavity of the recipient rat. The rat lung graft was rotated 180° along the vertical axis to achieve anatomic matching of right hilar structures. Another 10 consecutive rats had received orthotopic left lung transplantation as a control. ResultsAll lung transplantation procedures were technically successful without intraoperative failure. One rat (10%) died of full pulmonary atelectasis after right lung transplantation, whereas all rats survived after left lung transplantation. No significant difference was observed in heart-lung block retrieval (8.6 ± 0.8 vs 8.4 ± 0.9 minutes), cuff preparation (8.3 ± 0.9 vs 8.7 ± 0.9 minutes), or total procedure time (58.2 ± 2.6 vs 56.6 ± 2.1 minutes) between the right lung transplantation and standard left lung transplantation groups (P > .05), although the cold ischemia time (14.2 ± 0.9 vs 25.5 ± 1.7 minutes) and warm ischemia time (19.8 ± 1.5 vs 13.7 ± 1.8 minutes) were different (P < .001). ConclusionsRight lung transplantation with a left-to-right inverted anastomosis in a rat model is technically easy to master, expeditious, and reproducible. It can potentially imitate the principle of clinically inverted lung transplantation and become an alternative to standard left lung transplantation.

Viable Allogeneic Mitochondria Transplantation Improves Gas Exchange and Alveolar-Capillary Permeability in Rats with Endotoxin-Induced Acute Lung Injuries.

Background: Acute lung injuries (ALI) cause disruption of the alveolar-capillary barrier and is the leading cause of death in critically ill patients. This study tested the hypothesis that the administration of freshly isolated viable allogeneic mitochondria can prevent alveolar-capillary barrier injuries at the endothelial level, as mitochondrial dysfunction of the pulmonary endothelium is a critical aspect of ALI progression.Methods: ALI was induced by intratracheal lipopolysaccharide instillation (LPS, 1mg/kg) in anesthetized rats. Mitochondria (100 µg) were isolated from the freshly harvested soleus muscles of naïve rats and stained with a green fluorescence MitoTracker™ dyne. A mitochondria or placebo solution was randomly administered into the jugular veins of the rats at 2 h and 4 h after ALI induction. An arterial blood gas analysis was done 20 h later. The animals were then sacrificed and lung tissues were harvested for analysis.Results: An IVIS Spectrum imaging system was used to obtain ex vivo heart-lung block images and track the enhancement of MitoTracker™ fluorescence in the lungs. Mitochondria transplantation significantly improved arterial oxygen contents (PaO2 and SaO2) and reduced CO2 tension in rats with ALI. Animals with mitochondrial transplants had significantly higher ATP concentrations in their lung tissues. Allogeneic mitochondria transplantation preserved alveolar-capillary barrier function, as shown by a reduction in protein levels in the bronchoalveolar lavage fluid and decreased extravasated Evans blue dyne and hemoglobin content in lung tissues. In addition, relaxation responses to acetylcholine and eNOS expression were potentiated in injured pulmonary arteries and inflammatory cells infiltration into lung tissue was reduced following mitochondrial transplantation.Conclusions: Transplantation of viable mitochondria protects the integrity of endothelial lining of the alveolar-capillary barrier, thereby improving gas exchange during the acute stages of endotoxin-induced ALI. However, the long-term effects of mitochondrial transplantation on pulmonary function recovery after ALI requires further investigation.

Anatomical variations of bronchial distribution in the upper lobe of the right lung

Bronchial distribution in the upper lobe of the right lung is subject to many anatomical variations. The control of these variations is essential for the interpretation of endoscopic examinations. It also offers a better guarantee for safe surgery during segmentations in the right upper lobe. In this preliminary work, the operation of 30 heart-lung blocks treated by the injection-corrosion method, 100 CT chest and dissections of 3 resected specimen of right upper lobectomy, allowed us to study the bronchial distribution in the upper lobe of the right lung as well as their anatomical variations in melanoderm subjects. Our results were as follows: out of the 133 specimens, the right upper lobar bronchus was born on average 1.8 cm from the tracheal bifurcation, with an average length of 1.75 cm. There were 6 termination modes, dominated by bifurcations, which involved 52% of the specimens, and in 5 modes. Trifurcation accounted for 47.37% of cases. There was a case of quadrifurcation. These results allowed us to discuss anatomical variations of the bronchial shaft of the upper lobe of the right lung. Taking into account these variations will allow to avoid pitfalls and accidents during endoscopic examinations, medical imaging and pulmonary exeresis. KEY WORDS: Bronchi-Right Upper Lobe-Anatomy-Variations- Endoscopy-Imaging-Surgery.

Local lung coagulation post resection: an ex-vivo porcine model

Following non-anatomical resection of lung parenchyma with a Nd:YAG laser, a coagulated surface remains. As ventilation starts, air leakage may occur in this area. The aim of the present study was to investigate, whether additional coagulation either before or after ventilation has an additional sealing effect. Freshly slaughtered porcine heart-lung blocks were prepared. The trachea was connected to a ventilator. Using a Nd:YAG laser (wavelength: 1320 nm, power: 60 W), round lesions (1.5 cm in diameter) with a depth of 1.5 cm were applied to the lung using an 800-μm laser fiber (5 s per lesion). Group 1 (n = 12) was control. Additional coagulation was performed in group 2 (n = 12) without and in group 3 (n = 12) with ventilation restarted. Air leakage (ml) from the lesions was measured. The thickness of each coagulation layer was determined on histological slices. Differences between individual groups were analyzed by one-way ANOVA (significance p < 0.05). After resection, 26.2 ± 2.7 ml of air emerged from the lesions per single respiration in group 1. Air loss in group 2 was 24.6 ± 2.5 ml (p = 0.07) and in group 3 23.7 ± 1.8 ml (p = 0.0098). In comparison to groups 1 and 2 thickness of the coagulation layers in group 3 was significantly increased. After non-anatomical porcine lung resection with a Nd:YAG laser, additional coagulation of the ventilated resection area can reduce air leakage.

Contemporary Indications for Heart-Lung Transplantation

<h3>Purpose</h3> Currently, there are patients who meet criteria for lung transplant who are denied listing because of underlying heart disease. Likewise, there are patients who meet criteria for heart transplant but are denied listing because of lung disease. In addition, end-stage pulmonary hypertension has become more commonly treated with bilateral lung transplantation. From 2006 to 2018, no adult heart-lung transplantation (HLT) was performed in New York State. Given this unmet need our center has renewed interest in HLT. <h3>Methods</h3> All patients who underwent HLT from 06/2019 to 09/2020 were reviewed. Patients were listed per standard UNOS guidelines. The primary outcome was hospital and 1year survival. <h3>Results</h3> 10 patients (50% male; mean age 54±8 years) underwent HLT during this period. All patients had been deemed not appropriate candidates for isolated heart or lung transplant at more than one transplant center. The indication for HLT was interstitial lung disease (ILD) with ischemic cardiomyopathy (n=4), nonischemic cardiomyopathy with PAH (n=2), PAH with RV failure (n=2), and ILD with RV failure (n=2). Median lung allocation score and heart waitlist status were 71 (IQR: 41-85) and 2 (IQR: 1-3) respectively. Three patients had re-operative sternotomies. Two patients bridged to transplant with VA-ECMO, two with VV-ECMO and three patients required IABP. One patient received a DCD donor heart-lung block utilizing normothermic regional perfusion, and two patients received Hep C+ donor organs. One patient was left on central VA-ECMO postoperatively for early lung PGD. No lung PGD grade 3 at 72 hours or significant heart PGD at any time point were observed. At a median follow up of 287 (IQR: 108-401 days), all patients are alive at home. <h3>Conclusion</h3> Our initial center experience demonstrates that appropriately selected patients with end stage heart and lung disease who would otherwise die without transplantation can be offered HLT with excellent short-term outcomes.

The efficacy of ice recrystallization inhibitors in rat lung cryopreservation using a low cost technique for ex vivo subnormothermic lung perfusion

Although lung transplant remains the only option for patients with end-stage lung failure, short preservation times result in an inability to meet patient demand. Successful cryopreservation may ameliorate this problem; however, very little research has been performed on lung cryopreservation due to the inability to prevent ice nucleation or growth. Therefore, this research sought to characterize the efficacy of a small-molecule ice recrystallization inhibitor (IRI) for lung cryopreservation given its well-documented ability to control ice growth.Sprague-Dawley heart-lung blocks were perfused at room temperature using a syringe-pump. Cytotoxicity of the IRI was assessed through the subsequent perfusion with 0.4% (w/v) trypan blue followed by formalin-fixation. Ice control was assessed by freezing at a chamber rate of −5 °C/min to −20 °C and cryofixation using a low-temperature fixative. Post-thaw cell survival was determined by freezing at a chamber rate of −5 °C/min to −20 °C and thawing in a 37 °C water bath before formalin-fixation. In all cases, samples were paraffin-embedded, sliced, and stained with eosin.The IRI studied was found to be non-toxic, as cell membrane integrity following perfusion was not significantly different than controls (p = 0.9292). Alveolar ice grain size was significantly reduced by the addition of this IRI (p = 0.0096), and the addition of the IRI to DMSO significantly improved post-thaw cell membrane integrity when compared to controls treated with DMSO alone (p = 0.0034).The techniques described here provide a low-cost solution for rat ex vivo lung perfusion which demonstrated that the ice control and improved post-thaw cell survival afforded by IRI-use warrants further study.

Transplantation of viable mitochondria improves right ventricular performance and pulmonary artery remodeling in rats with pulmonary arterial hypertension

ObjectiveBecause mitochondrial dysfunction is a key factor in the progression of pulmonary hypertension, this study tested the hypothesis that transplantation of exogenous viable mitochondria can reverse pulmonary artery remodeling and restore right ventricular performance in pulmonary hypertension. MethodsPulmonary hypertension was induced by parenteral injection of monocrotaline (60 mg/kg) and creation of a left-to-right shunt aortocaval fistula in rats. Three weeks after creation of fistula, the animals were randomly assigned to receive intravenous delivery of placebo solution or allogeneic mitochondria once weekly for 3 consecutive weeks. Mitochondria (100 μg) were isolated from the freshly harvested soleus muscles of naïve rats. Transthoracic echocardiography was performed at 3 weeks after mitochondrial delivery. ResultsEx vivo heart-lung block images acquired by an IVIS Spectrum (PerkinElmer, Waltham, Mass) imaging system confirmed the enhancement of MitoTracker (Invitrogen, Carlsbad, Calif) fluorescence in the pulmonary arteries. Mitochondria transplantation significantly increased lung tissue adenosine triphosphate concentrations and improved right ventricular performance, as evidenced by a reduction in serum levels of B-type natriuretic peptide and ventricular diameter. Right ventricular mass and wall thickness were restored in the mitochondrial group. In the pulmonary arteries of rats that received mitochondrial treatment, vascular smooth muscle cells expressed higher levels of α-smooth muscle actin and smooth muscle myosin heavy chain II, indicating the maintenance of the nonproliferative, contractile phenotype. The hyper-reactivity of isolated pulmonary arteries to α-adrenergic stimulation was also attenuated after mitochondrial transplantation. ConclusionsTransplantation of viable mitochondria can restore the contractile phenotype and vasoreactivity of the pulmonary artery, thereby reducing the afterload and right ventricular remodeling in rats with established pulmonary hypertension. The improvement in overall right ventricular performance suggests that mitochondrial transplantation can be a revolutionary clinical therapeutic option for the management of pulmonary hypertension.

Cyclosporin A Administration During Ex Vivo Lung Perfusion Preserves Lung Grafts in Rat Transplant Model.

Despite the benefits of ex vivo lung perfusion (EVLP) such as lung reconditioning, preservation, and evaluation before transplantation, deleterious effects, including activation of proinflammatory cascades and alteration of metabolic profiles have been reported. Although patient outcomes have been favorable, further studies addressing optimal conditions are warranted. In this study, we investigated the role of the immunosuppressant drug cyclosporine A (CyA) in preserving mitochondrial function and subsequently preventing proinflammatory changes in lung grafts during EVLP. Using rat heart-lung blocks after 1-hour cold preservation, an acellular normothermic EVLP system was established for 4 hours. CyA was added into perfusate at a final concentration of 1 μM. The evaluation included lung graft function, lung compliance, and pulmonary vascular resistance as well as biochemical marker measurement in the perfusate at multiple time points. After EVLP, single orthotopic lung transplantation was performed, and the grafts were assessed 2 hours after reperfusion. Lung grafts on EVLP with CyA exhibited significantly better functional and physiological parameters as compared with those without CyA treatment. CyA administration attenuated proinflammatory changes and prohibited glucose consumption during EVLP through mitigating mitochondrial dysfunction in lung grafts. CyA-preconditioned lungs showed better posttransplant lung early graft function and less inflammatory events compared with control. During EVLP, CyA administration can have a preconditioning effect through both its anti-inflammatory and mitochondrial protective properties, leading to improved lung graft preservation, which may result in enhanced graft quality after transplantation.

Rat lung transplantation model: modifications of the cuff technique.

BackgroundAlthough the cuff technique in rat lung transplantation (LTx) has a long history, it remains technically challenging. We have developed key tricks and modifications in the devices and the cuff technique that optimize the rat LTx model to achieve successful operations during a short learning period.MethodsAltogether, 180 consecutive rats underwent orthotopic left LTx performed by a single surgeon using our modified devices and procedures. Allogeneic and syngeneic transplantation were performed using Lewis rats as recipients and Brown Norway and Lewis rats as donors. Allogeneic recipients were treated with cyclosporine during the first week. Recipients were sacrificed at various time points after ≥2 weeks.ResultsA special cuff-preparation plate was created using a petri dish and two foam blocks. This modified plate stabilizes the preparation and prevents donor lung compression. A “┴”-shaped incision was carved into the front wall of the pulmonary artery (PA) using micro-scissors. “V”-shaped incisions were made from the inferior-to-superior branches of the pulmonary vein (PV) and bronchus. A “pendulum model” was applied at implantation to make the hilar anastomosis tension-free and technically easier to perform. There were no intraoperative complications. Ten rats (5.6%) experienced partial or full pulmonary atelectasis. Five deaths (2.8%) due to pleural effusion occurred during the follow-up period. The operative times for heart-lung block retrieval, cuff preparation, cold ischemia, warm ischemia, and total procedure time were 8.4±0.8, 11.6±1.5, 25.1±2.2, 8.1±1.2, and 46.7±2.8 min, respectively.ConclusionsThe key tricks and improvements we made in the cuff technique for rat LTx provided the advantages of expeditiousness, a low complication rate, and a high success rate.

Video of donor heart-lung block retrieval (time to accomplish it is twice as fast as in the past)

Video of donor heart-lung block retrieval (time to accomplish it is twice as fast as in the past)

Lung Transplantation: The Indian Experience and Suggested Guidelines Part II A: The Technique of Lung Transplantation

Background: A series of lectures and workshop on lung and heart–lung transplantation were organized by the Departments of Cardiology and Cardiothoracic Surgery at All India Institute of Medical Sciences, New Delhi, and the Department of Heart and Lung Transplantation at Apollo Hospitals, Chennai. The 1st day workshop on preoperative workup part was published as Part-I in this journal in August 2018. The hands-on cadaveric workshop conducted on May 20, 2018, forms the basis of the first section of the second part (Part II a) which deals with the technique of lung transplantation. The techniques of lung transplantation and Indian scenario are also reviewed. Methodology: The technique practiced by the transplant team at Apollo Hospitals, Chennai, was used for the demonstration of donor lung, heart–lung block harvest and implanting lungs, heart–lung block, while also discussing other possible techniques. Human cadavers were used, and live high-definition audio–video transmission to the lecture hall was made. Results: Accurate figures regarding lung transplantation in India are still not available. However, as per the Indian Society for Heart and Lung Transplantation, current data suggest that overall, about 1050 heart transplants and 310 lung transplants have been done in India. Of these, it is anticipated that national data regarding number of single-lung transplants, double-lung transplants, and heart–lung transplants will be available soon. The Department of Heart and Lung Transplantation at Apollo Hospitals, Chennai, has performed a total of 69 hearts and 149 lungs in 119 patients in various combinations: isolated heart transplants (40 patients), double-lung transplantation (DLT) (43 patients), single-lung transplantation (7 patients), heart and DLT (27 patients), en bloc heart and liver transplantation (1 patient), and combined heart–lung and kidney transplantation (1 patient). While the survival data for India are not currently available, our 3-year survival for DLT is 76.2%. Conclusions: While the surgical technique demonstrated is used in most of our cases, at times, different techniques have had to be adopted based on challenges confronted on the operating table. However, we have found that developing surgical protocols and maintaining consistency in the operative techniques translates into good surgical outcomes.