Dr. M. Judah Folkman Research Day - Dr. Savas Tsikis, Dr. Kamila Moskowitzova, Julia Clarke, and Dr. Victor Alemany

Good morning, everybody. I'd like to welcome you to the 24th annual, well, now it's called the M. Judith Falkman Research Day. This year we received 187 abstracts, and every year the numbers keep going up and we break a record every year. As a result, we really had to increase the number of judges so that everybody has a modest amount of extra work to do to evaluate all the abstracts that were submitted. And from that group, we've picked 12 presentations for this morning. Four will be at the medical grant rounds this afternoon and four will be at the afternoon session at 330 to 430. Although we've picked these 12, everybody who submitted an abstract is a winner. I, you know, when reading through all the abstracts every year, I'm just amazed that the outstanding work that's been done by the research community here at Children's. It's fantastic, and I love coming to coming in the door every day because I learned something new. I'd like to introduce Bernard, a method of promote, who's taken over from Jordan Creepberg and now director of the Office of postdoctoral affairs. And he will introduce a couple of the abstracts later this morning. And I'd like to also thank Lou Ann Posey, who couldn't be here today. She is in the helmsman who has steered our ship into port without any grounding or mishaps at all. She has some personal issues with family and couldn't be here today, but I wanted to acknowledge all the work that she did. So, and we would like to thank the medical staff organization in the Office of postdoctoral affairs for their generous contributions and willingness to work with us to have this day. So without further ado, I'd like to introduce service, it's a kiss, who will present direct thrombin inhibitors as alternatives to happen to preserve long growth function and a realization in a hearing model of compendium. Of compendium compensatory long growth. Thank you. We would like to thank the organizing review committee for the opportunity to present our work. We have no relevant financial disclosures. In general, they're from out of cornea or CD8s arises from a congenital defect in the diaphragm, at least to the herniation of abdominal organs into the thoracic cavity in utero. The incidence is approximately one to four cases for 10,000 like births. As a result of this in uter event, infants are frequently born with severe bilateral pulmonary hypoplasia, pulmonary hypertension and cardiac dysfunction. The cardiopulmonary disturbances can be so severe that infants require extra corporeal membrane oxygenation or ECMO for the survival. While on ECMO, into maintained circuit pedancy, systemic interquagulation is necessary, with Heparin being the most common choice of anticoagulant use in a setting. The direct thromod inhibitors abbreviated DTIs, bivalerdin and argatriban, are alternatives for this indication. Unfortunately, even with the use of ECMO, the survival rate for CD8s patients who require it remains at a dismal 50%. Furthermore, survival in part is a race against time for lung growth and regeneration to occur, as mortality increases with each additional day on ECMO. Our lab employs a CD8 model of complementary lung growth or CLG. In this model, mice are anesthetized, intubated, and the less lung is removed via less thoracotomy and hyalurlication. The remaining right lung is left in situ, and its growth is studied at various postoperative time points. In this model, we previously demonstrated that Heparin emperors both lung growth and function. In particular, at postoperative day 4, following left nomenectomy, Heparin mice demonstrated impaired pulmonary mechanics, decreased respiratory volumes, and reduced exercise tolerance. The mechanism is thought to involve impaired angiogenesis, whereby Heparin was shown to inhibit the vascular and dothelial growth factor or vegetative pathway by interaction with the extracellular matrix. We hypothesized that the direct throbinin inhibitors by Valeridin or Gatreban, owing to their more specific mechanism of action, will preserve lung growth, function, and angiogenic signaling in this model. To evaluate the Cypopsis, we performed both in vitro and in vivo experiments. In the in vitro experiments, human lung and dothelial cells were plated at 30% confluence in gelatin-coated wells. It was also thought to be a starved overnight and following overnight's survey were treated with increasing concentrations of Heparin, Bivaleridin, or Gatreban. Following a 72-hour incubation period, both cell proliferation and apoptosis were assessed using appropriate commercial assays. For the in vivo experiments, C57-Black-16 mice underwent left nomenectomy, followed by subcutaneous implantation of osmotic pumps, which in this experiment allowed for continuous anticoagulation to be achieved, thereby simulating the clinical scenario of interest that is in vivo. Animals were randomized to a total of four groups, a control group where animals received osmotic pumps that were loaded with normal saline, and three anticoagulated groups where mice were continuously and therapeutically anticoagulated via osmotic pump with either Bivaleridin or Gatreban or Heparin. Initial dose response studies were performed to determine the concentration and rate of drug delivery that was necessary to achieve therapeutic anticoagulation, and this was confirmed by PTT measurement. All outcomes were assessed on post-operative day 8, as this is a time-pointed with compensatory lung growth is known to be complete. The main outcome measures for the in vivo experiment included assessment of lung growth, histology and more prometric analysis, immunofluorescence, treadmill exercise tolerance testing or TETs, and mechanistic analysis through lung protein and RNA sequencing. For the in vitro experiments, Heparin decreased lung and ethereal cell proliferation, starting at the 0.5 unit per ml dose, which is representative of the in vivo concentration of Heparin following low dose sub-syrputic Heparin treatment. By Vivaleridin or Gatreban, however, did not significantly impact on ethereal cell proliferation at all doses such as examined. In terms of the in vitro apoptosis assay, Heparin significantly increased in ethereal cell apoptosis in a dose-dependent fashion. Interestingly, by Vivaleridin consistently inhibited apoptosis at all doses examined, suggesting a protective pro-indithereal effect. Our Gatreban for the most part did not impact cell apoptosis. With these experiments, we turned our attention to the in vivo experimentation. Lung growth was assessed by lung volume measurements through the water displacement method. In this method, the remaining right lung is carefully dissected off of the chest wall after euthanasia and inflated in 10% formalin at a constant barometric pressure of 35 cm water. The formalin inflated lung is then weighed in the water contained beaker. With this method, Heparin is mice demonstrated significantly decreased lung volume compared to saline treated controls. Lung growth was not significantly affected with treatment with either Vivaleridin or Gatreban, as indicated by the lack of a significant difference compared to controls. To determine if all of these effects correlate, it's a further characterized, the effects of anticoagulation on lung growth, more for metric analysis of stained lung sections was performed by a mass observer based on the principles of seriology. Based on this analysis, Heparin mice demonstrated decreased avialization as a composite outcome of perencomal volume, Avialer volume, septal surface area, which is the area available for gas exchange, and finally Avialer density. By Vivaleridin or Gatreban, however, for the most part did not impact the composite outcome of avialization. These findings are further corroborated by representative hematoccylin and inelocent stained lung sections, whereby the Heparin mice demonstrate decreased avialization compared to controls. While the Vivaleridin or Gatreban treated mice had preserved avialization. We then performed, we then assessed lung vasculization by immunostain for the endothelial cell marker CD31. CD31 staining, which is represented by green on the screen, was decreased in Heparin mice compared to controls, as demonstrated by the representative immunoclerusin stained sections in panel A, and the quantified analysis of signal intensity in panel B. By Vivaleridin or Gatreban, however, did not significantly impact and CD31 staining and thereby by extension long vasculization. The determinant of all of these outcomes correlated with a physiologically relevant outcome, a separate cohort of mice underwent treadmill exercise tolerance testing at baseline prior to surgery, followed by repeat testing prior to euthanasia, and the percent change in exercise performance was determined for each individual mouse. This allows for each mouse effectively serves to effectively serve as its own control, thereby counting for intrinsic behavioral differences between animals. In this experiment, Heparin significantly decreased exercise performance, as measured by the decreased percent change in distance run compared to controls, as well as time spent running. By Vivaleridin or Gatreban, however, did not significantly impact exercise performance based on these metrics. The further understand the impact of the different anticoagulants on lung growth, we turned our attention to a very important pathway involved in endrogens and pro lung growth, the VEGF R2 pathway. In this pathway, VEGF finds to its key receptor VEGF R2 leading to its intracellular activation through phosphorylation. As a result of the phosphorylation, a number of downstream signaling cascades ensue that leads to increased cell proliferation, vascular permeability, cell migration, and cell survival. To evaluate this pathway, protein was extracted from lung tissue and used for Western immunoblodding of these various targets of interest. Represented immunoblots are shown in panel A. VEGF was significantly upregulated by Vivaleridin treated mice, which considering the in vitro experiments demonstrating an anti-apoptotic effect is a particularly interesting result. Our Gatreban and Heparin did not significantly impact VEGF expression. However, activation of that key receptor VEGF R2 was significantly decreased in Heparinized mice, as measured by the ratio of the phosphorylated or active form of the receptor to the total form of the receptor. VEGF R2 activity was not significantly impacted by anticoagulation with either Vivaleridin or our Gatreban. To further characterize the effects of Heparin in particular on growth factor pathways more broadly and characterize its mechanism of inhibition, we then performed BORK RNA sequencing of lung tissue of Heparinized mice and compared them to normal saline treated controls. Ingenuity pathway analysis demonstrated broad down regulation represented by green in the screen of key canonical growth factor pathways that are implicated in alveolar genesis, cell proliferation, and embryonic proliferation. This resulted in a projected increase of lung hypotasia and it consistently matches both the results of the in vitro as well as in Vival experiments. In conclusion, in vitro Heparin inhibited lung and ethereal cell proliferation and increased apoptosis. The continuous administration of Heparin in Vival impaired lung growth, alveolarization, vascularization, and exercise performance. On mechanistic analysis, protein immunoblodding confirmed that Heparin inhibited angiogenic signaling and RNA-C offered additional insight onto the impact of Heparin on growth factor pathways more broadly. Importantly, by Vivaleridin and our Gatreban did not impact these key outcome measures either in the in vitro or in vivo experimentation. Based on these results, this supports the use of direct-throwman inhibitors for systemic antipagulation on ECMO in CDH patients clinically who depend on expeditious lung growth for their survival. Based in part on the data, some centers including our own institution are now transitioning to the use of Vivaleridin for their circuits. Ultimately, clinical studies on the impact of Heparin compared to DTIs on CDH outcomes for Warrington. I would like to thank our study team, acknowledge their funding sources, and I'll be happy to take any questions. Are there any questions? I'm sorry. We have not examined that yet, but it would definitely be interesting to look at. Yeah, that's so that's an interesting question. I did that. I dealt with the literature following the in vitro experiments where Vivaleridin was shown to reverse the inhibition of endocelial cellapoptosis. And some papers that I found suggested that by directly inhibiting throbin, Vivaleridin is able to reverse the hyperprenubility of endocelial cells and therefore improve their ability to survive, which potentially is why we're seeing a antibiotic effect in the in vitro experiments. And so in terms of how that translates to more granularity in terms of the mechanism that's still an area of active investigation because it is a finding that we've consistently demonstrated time and time again, whereby after you treat human microvascular lung into philisos with Vivaleridin, you get deeply safe off doses. And there's different experiments in mind that you could determine that more broadly is that you can remove the media does contain some problems. So you can remove the trauma completely from the media and see if you're still seeing that effect because if you do still see that effect, and it suggests that by Vivaleridin is interacting with some other receptor or some other pathway to have that protective effect rather than meeting the proven so those experiments are currently ongoing. Thank you very much. The next presentation is from Camela, Moscow, Moscow, it's over trans and the anach fetal mRNA therapy, a protein novel strategy for pulmonary surfactant replacement and for the prevention of cystic fibrosis associated maconium helios. Thank you very much. Are you going to? Sorry, I can't just. Oh, no, yeah, I cannot do it from here and I don't see here. Sorry. Okay, good morning and thank you for the opportunity to present our work. We have no disclosures. Amniotic fluid is typically dismissed as the mere byproduct of basic physiologic processes that could only be considered for crude fetal therapies based on fetal swallowing and or aspiration of the amniotic fluid. However, it is much more than that. The interface between the fluid, the gestational membranes and the placenta is a complex and the dynamic structure that allows for transport of variety of biological agents. Two and from the fluid eventually reaching fluid fetus. This actually renders amniotic fluid to be one of the least invasive windows to the fetal circulation. As a direct result of that, our group has described diverse trans amniotic fetal therapies for variety of diseases. For example, trans amniotic immunos and stem cell therapies in which respectively immunoglobulins or stem cells are delivered to the fetus via this practically minimally invasive window. The latest addition to these strategies has been trans amniotic nucleic acid therapy or train it initially attempted with mRNA as a therapeutic agent. However, prior study using mRNA encoding for Luciferase, we have shown that encapsulated mRNA delivered to the amniotic fluid can also be transported through this amnioplasental interface and reach the fetal circulation besides being just simply swallowed or aspirated. In the present study, we tested for the first time a potential therapeutic application of trans amniotic mRNA therapies, more specifically involving the replacement of pulmonary surfactant protein and cystic fibrosigants, transplant membrane conducted regulator or CFTR. Pulmonary surfactant is a complex lipoprotein mixture produced by type two cells in the lungs. By lining the epithelium at the air fluid interface, it allows either a usage surface tension and allows for adequate gas exchange. Lipids especially phospholipids constitute the majority of the surfactant, yet proteins play an important role in surfactant function. By stabilizing the mixture and facilitating the lipid absorption to the alveolar surface, therefore creating active surface monolayer. Although force surfactant protein, surfactant protein V or SPB is one of the most important ones. Surfactant simply becomes ineffective in absence of SPB. It's important is further reinforced by the fact that genetic mutation in a gene for SPB can cause severe neonatal distress. The prime example of this is SPB deficiency, which is congenital monogenic autosumol recessive disease causing respiratory failure despite exogenous surfactant administration. And the only curative treatment is line transplantation in early lives. Much more common respiratory distress of pre-maternity is also directly linked to surfactant deficiency, including SPB. Curent surfactant replacement therapies do not exactly resemble human surfactant, and there is still notable mortality and morbidity. Another disease which can impact neonates in terinatal period is a cystic fibrosis. This is monogenic autosumol recessive disease, in which mutation of CFTR, protein which serves as a chloride channel occurs. Mutation of CFTR cause impaired chloride epilogue through the channel, eventually leading to fig mucous buildup on a top of various mucous surfaces. Besides malfunction, certain mutation may result in CFTR either being misfolded and unable to reach the cell apical membrane or not even being transcribed at all. Either scenario can also lead to fig mucous buildup in various organ systems. In a perinatal period, this mucous plaque can result in intestinal obstruction, leading to meconium elials. So in our study, we saw to determine whether exogenous human SPB and CFTR mRNAs could be incorporated and translated by the fetal lung and incents intestine respectively after simple intra-amnotic injection initially in a healthy red model. And if so, this could become a novel strategy for pulmonary surfactant protein replacement and for prevention of CF associated meconium elials. A commercially available human SPB and human CFTR mRNA were obtained and encapsulated by self-assembling lipid and polymer composite into lipopolyplex. The encapsulation efficiency was assessed by fluorometric essay. On justationally 17 or E-17, fetuses and event intra-amnotic injection of either mRNA and encapsulated in a lipopolyplex or lipopolyplex only without any mRNA which served as the control. In SPB group, then daily until term, fetal lungs were analyzed for presence of human SPB by Eliza. The postfatidil colline levels, as the surrogate for overall surfactant production, were measured in amniotics fluid. In CFTR group, similarly, fetal intestines was collected and evaluated by Eliza for presence of human CFTR and under an immunofluorescence staining for human CFTR. Samples from control animals were obtained at the at-term only at E-20-M. A encapsulation rate for both mRNA was very high, which is similarly to what we and others have reported for other mRNA products. In SPB group, fetuses receiving human SPB mRNA showed improved viability at two different time points at E-18 and E-20. In CFTR group, no significant difference between the groups or time point was observed. Overall, fetal viability was 85.3%. No maternal mortality was observed overall. When controlled by mRNA-3 injection, human SPB was present in a fetal lung at E-18 in 19 and E-21. There was a significant increase overall over time in the levels of SPB, which still high levels at term. As you may have noticed, there is positivity in the controls animals as well, which might suggest some degree of human rat homology for SPB, at least at this anatomical location. Fetitial calling levels tended higher in mRNA group compared to control at term. However, this did not reach significance. This is perhaps not surprising given the use of healthy model. In CFTR group, human CFTR levels were significantly increased at each time point after inter-anniotic injection, with increasing trend over first three days and still high levels at term. Immunofluorescence staining showed a high strong signal at the apical membrane of the entero side, which is exactly the location of the functional CFTR protein. And control animals did not show any signal on immunofluorescence. In conclusion, encapsulated exogenous mRNA encoding for human SPB and CFTR can be incorporated and translated at the clinically relevant sites, following simple inter-anniotic injection in healthy rat model. Trans-anniotic mRNA therapy could become a novel strategy for pomenars affecting protein replacement and for prevention of CF associated meconium elions. I'm grateful to all my co-authors, to Department of Surgery and to you for your attention. Thank you. I have a question for you. Do you think how early in the course of identifying children with CSF? Could it be a good idea to inject the SPB or CSF? Yeah, so there are two possible applications that we are thinking of. Initially, as I mentioned, is prematurity, which would be in the children that are born prematurely or in the genetic disease, which is SPB deficiency, which that can be done by screening of the babies through the peripheral blood of the baby. The DNA is screened for the presence of abnormal fetal DNA. So upon finding a presence of genetic mutation in SPB in a fetus, that could be the time that the fetuses could be considered for this therapy. There are questions for noise. There's a microphone. Sometimes in perinatal period, usually it's less than 10% of all CFDRC. Sometimes you can see it in prenatal ultrasound as there are like findings on the ultrasound that can suggest obstruction. In the settings of known CFTR for the babies, you might assume that that can be meconium elions. The next presentation is by Julia Clark, effective just in time rapid cycle deliberate practice training in the operating room, a valuable tool for enhancing components in pediatric intubation skills for novice anesthesia trainees. Thank you. Thank you so much. Okay, good morning, everyone. My name is Julia Clark. I am a research assistant with the park team in the NSD department. Thank you for the opportunity to present our abstract this morning. So pediatric intubation skills are crucial for trainees to master, especially in more high risk age groups. And so a dilemma that's faced in pediatric anesthesia is striking a balance between ensuring that novice trainees obtain firsthand experience, and also providing neonates and infants while also preserving patient safety. And so the just in time rapid cycle, deliberate practice training model aims to bridge this experience gap by providing trainees with supervised skill practice in a controlled simulation environment, potentially improving training competence and ultimately reducing intubation attempts and patients. We've been a handful of studies that are have tried to assess similar questions, particularly interesting study Miller at all conducted a secondary analysis of a randomized control trial conducted to determine the effect of using pre recorded videos of patient and the tracheal intubations on intubation performance. And so their control group received standard curriculum with bold and text and still images while their interventional group received the same standard curriculum, but also had nine videos of live intubations integrated throughout this curriculum. And each group also received simulation instruction after two didactic teaching sessions. So this study measured intubation success in two different ways. The first was time to successful intubation on four airway simulations and this study defined time to successful intubation as cumulative time to placing the endotracheal tube correctly regardless of intubation attempts. So we used a 14 item knowledge base assessment and this was given before the first didactic teaching session and again after all of the teaching sessions were completed. Trainees were also given a survey assessing intubation competence both before and after their curriculum. And then the first time Miller at all found was that self reported confidence increased across all participants after this curriculum. However, pre curriculum confidence did not correlate with trainee performance on either the knowledge base assessment or the simulation assessments. And they also found that there was no correlation between performance on the knowledge and simulation assessments and post curriculum confidence. They also saw no no correlation between total time to successful intubation and post curriculum confidence. So you know, it makes the question, you know, where do we go from here? A lot of questions still remain, particularly how to you know continue to strike that balance between increasing patient safety and also allowing these novice trainees to learn these intubation skills. So our project comes in and our project we observe neonatal and infant intubations performed by novice anesthesia trainees to explore whether the stress and time rapid cycles a little bit practice training model improves true and perceived measures of competence and intubation skills. This was an IRB approved non cross over prospective randomized control trial. And then we have a pediatric anesthesia trainees at their hospital orientation. And once these trainees were enrolled, they were block randomized into an experimental or control group. And in our study, our experimental group received a just in time rabbit cycle, deliberate practice simulation session with one of our anesthesia airway coaches within one hour of their live intubation case. It was aimed to observe up to five consecutive distinct infant intubations per trainee and the outcomes we were assessing in each group were core maclachane views as objective measures of competence and trainee survey responses as subjective measures of competence. Infants who were less than or equal to 12 months of age with an ASA of one to three who were having a pre planned and detritual intubation. And we excluded those cases where patients had a known or suspected difficult airway or intubation had a complex congenital heart disease any kind of oxygen requirement or artificial airway as well as a known COVID-19 infection. So our recruitment occurred from August 1st, 2020 to April 30th, 2022. We enrolled 150 trainees, 70 of those in the experimental group and 83 of those in the control group. And overall, we observed 515 intubations for this project, 232 in the experimental group and 283 in the control group. And what we saw that was across all intubation rounds, the experimental group reported significantly more grade one views on first intubation attempts for both direct laranoscopy and video assisted direct laranoscopy. In addition, while we saw there was no significant difference between study groups in the pre study competency surveys, we saw that experimental trainees reported significantly higher levels of perceived competence compared to control trainees across all post study survey questions. So in conclusion, this just in time rapid cycle deliberate practice training led to an increased frequency of grade one views during first intubation attempts with both DL and VADL techniques. And this training model improved trainee competence, trainee perceived competence in their intubation skills. However, for the researchers encouraged to explore how and if these two competency domains are related and to evaluate other ways in which this training model man hands competence and novice trainees. So I'd like to thank the anesthesia department, Dr. Flynn, Covatzis, Park, Payton and sign the park team and the main OR nursing team and staff for their help in completing this project. And I'll take any questions if anyone has them. I didn't realize you were doing it. These are anesthesia residents on their first rotation here. So these are in effect anesthesia residents getting their first exposure to pediatric laranoscopy. Correct. So we actually enrolled residents, fellows, NS RNAs who were rotating in the main OR. And once a trainee was enrolled in the project, if they come back for a second rotation here at the hospital, he did not involve them again. All right. Did you look at how long it took to successful intubation in the two groups? We took a couple of different time points and that varied based on we took a total time to successful intubation and then we also measured times in between each intubation attempt and we also tallied the traces of the two events of the two adults that the two were doing to help them. And so we kind of delineated those into each different airway attempt. We defined, kind of one attempt as you see the cords you pass the tube, if you have to pull the two back out of the mouth and try again, that's one attempt. actually had a couple of abstracts for this project because we just took so much data, but we did see it do some time analyses as well. We just are getting a bit more into those as we write our manuscripts. Excellent. The presentation. I was concerned with the background Miller study that you presented where it demonstrated that after education there was increased confidence, confidence, without increased competence. And your study, which had just didn't say what competence was, Dr. Bauer sort of alluded that maybe there's more abstracts, you also showed increased confidence, but it sounds like it has some data. Can you give us a sneak preview on whether there's increased competence? So that is a question that when we were kind of starting to sift through our data and kind of see how we wanted to take these abstracts, what the difference is between confidence and competence? And I think that is sometimes a bit of a subjective question. What we tried to look at was so, how we tried to measure confidence was through that pre-imposed study survey. I can't really speak to trainee confidence. We're still sorting through all the data that we were able to collect, but definitely increased trainee competence. Although we had the experimental trainees got an additional kind of a dendum to those pre-imposed study survey questions where they had some kind of free response open text where they could write in some comments about their time in the project and things like that. And a lot of them remarks that they did feel much better after having even just one simulation training. Every experimental trainee received a coaching session before each intubation they did for the study. But a lot of them said that even just after one or two, they felt much better and more confident in their training techniques. And we had about four or five different airway coaches who all kind of gave different feedback. And so a lot of the experimental trainees wished that everyone could have the opportunity to have those coaching sessions. And maybe it's not necessarily required to have five consecutive ones, but one or two live or freshers before you go into that more hectic environment and do those things. Definitely, I think, you know, made them feel a lot better. So, you know, there's a lot of room to make some judgments there, but, you know, I think we'll wait to see what our manuscripts have. So now that I have seen Dr. Bauer introduce the previous speakers, I have a lot more confidence to introduce the last speaker, which is Dr. Victor Alimani. And he's going to talk about mitochondrial transplantation in pediatric and neonatal porousine hearts in a model of DCD heart transplantation. Good morning, everyone. Thank you for the committee for having me today. This is my disclosure slide. Our transplant is the preferred treatment strategy for patient suffering from medical irrefactory and stage heart failure. At present, most of the donor hearts used for transplantation in the USA are obtained from donation after brain death or DVD. This approach has proven effective in the last decades, but the number of donor hearts remains significantly lower than that required to meet the demands. Donation after circulatory death or DCD has been proposed as alternative to increasing the pool of donors. The difference between a DCD and a DVD is that a DCD patient also has an uncurable terminal illness, but doesn't meet the criteria for DVD. It's chosen by either by his life, living will or by his family to be withdrawn from life support. And there's the team has to wait for should go to arrest to happen. And then there's a standby period that bars depending on the country. And that's the main problem of DCD donation. It has a warm ischemia time that DVD doesn't have. In this study, we investigated the efficacy of mitochondrial transplantation to improve the viability and function of neonatal and piegetic DCD hearts donation. For this purpose, we have used two groups of drug-shyte pigs, neonatal and piegetic whose weight and harrowate are consistent between our mitochondria group, hearing red and the big ol' group in black. And I agree with you, and according to the literature, to a baby two to a month of age in the case of the neonatal group and an age of two to eight years old in the case of the pediatric group. This is our protocol. And in the first part of the experiment is similar in every group. We perform a mid-line tsunami, dissect the IVC and as we see perform an epicardial echocardiogram to ensure a good baseline function and valve competency. In the Shang group or our DVD group, the heart is arrested with the needle echocardiplija and harvested. And it's not exposed to any warm ischemia time that mimicking that way a DVD heart transplant. In our experimental branch of the study, in the case of our DCD hearts, we follow a very similar process to our regular DCD heart transplant. The animals we drone from life support, we wait for the map to equal the CBP pressures to equalize. And after a note-toge period of two minutes, we declare death. After exactly 20 minutes of warming ischemia time, we perfuse the Neocardiplija and the 10 minutes of cold ischemia time starts at that point. For the heart street with mitochondria transplantation, before we join the animal from life support, we harvest a piece of pectoralis muscle for mitochondrial isolation. M mitochondria were counted and by ability to wasn't sure previously to the mitochondria transplantation that we performed. During those 10 minutes of cold ischemia time, after the needle echocardiplija, we finish placing the canolas and place the balloon in the left ventricle to be able to measure the left ventricle developed pressure later on in the process. With this methodology, we are encountering very similar results to where is described in the human DCD heart transplants. The time to know flow, which is the time from withdrawal of life support to when the map equal to the CBP in our group is, as you can see, 6.83 minutes for the neonatal group and 6.73 minutes in the pediatric group, which is very similar and there's no significant difference with the 10 minutes found in human DCD groups. We use 20 minutes of warmest total warm ischemia time, because it's very similar to what it's been experimented and what it's been obtained in the DCD humans. After those 10 minutes of cold ischemia time that I was talking about, the heart is hung in the X-bibber perfusion system and is perfused for a total of 4 hours. In those 4 hours, the first 2 hours, as you can see in the graphic, are downloaded phase, simulating transport in the organ care system machine or the OCS machine or standby time. And during this time, after the first 15 minutes is when we inject the vehicle solution or the vehicle solution contain madocondria to the heart. The last 2 hours of the perfusion is the loaded phase, where an endiastolic pressure of sand to 9 millimeters of mercury is applied to the left ventricle to simulate loaded conditions inside or once the heart is transplanted. The excited perfusion machine is a system very similar to the OCS machine where we perfuse the heart to the Arctic Anola, assuming a competent, Arctic bath, which we had checked before previously with the echocardiogram. The blood flows to the conaries, into the cornice sinus, into the right atrium, into the right ventricle, and comes back to the reservoir through the canola that we place in the pulmonary artery. From the reservoir, it gets pumped again into the oxygenator and the heat exchanger. And from there, it goes back through the Arctic Anola into the heart. During the loaded phase, as you can see in the left, the arterial blood splits into the left atrium-compliance chamber to maintain a constant pressure of sand to 9 millimeters of mercury and to the Arctic compliance chamber to keep a steady pressure of 50 to 60 millimeters of mercury. During the reprusion, the first thing we can appreciate is the visual assessment, as you can see here, is the behavioral of the heart. First, when we repruse the heart, and after it achieves sinus rhythm, we can appreciate the baseline of the heart has a weak contraction after 15 minutes at the sign point, we inject the mitochondria through the pressure line pouring the Arctic Anola, and within 10 to 15 minutes, we can clearly appreciate chronic tropic and inotropic response in your video on the left. During the entire four hours of reprusion, we strive to keep some variable constant. In these graphics, you can appreciate the arterial PO2, hemoglobin, lactate, and Arctic blood pressure. In blue, we have the DVD heart or sham. In red is our treatment group, or DCD heart, streeted with mitochondria transplantation. And in black, you can see the DCD heart, streeted only with the vehicle solution. We can see that in both groups, for the neonatal amphibrated groups, we keep the arterial PO2 constant at 200 millimetre mercury, the hemoglobin constant, and 10 milligrams per deciliter, and the Arctic group pressure at a constant 55 to 60 millimetre mercury. We can see a slightly higher lactating in the neonatal group, which is due to the lower pumbrate, due to a lower connivable flow in this group, giving it a smaller size. Other variables that we try to keep constant through the profusion are the electrolytes, potassium, sodium, and calcium, in all groups, in both the neonatal and periodic hearts. For the calcium, as we, as we did in Beratur, the pH, and the PCO2, we followed the magnitude of a guidelines, trying to start with a slightly hypothetical semi-hyperthermic and associated perforated, with a calcium around 0.5 to 0.7, and increase lowly to the first 15 minutes to 1.2. One of the most significant results that we obtained during the two hours of loaded profusion is the lab ventricular function. In this graphic, we can see how the DCD-HARS-3 with myocondria in red achieve an equal or better lab ventricle developed pressure than the brain donor hearts in blue. At every time point during the profusion, there's statistical significant difference between the mitochondria group and the vehicle group, as you can see in the graphic. We find a very similar result on the left when we look at the fractional shortening with the Picardial Cochardiography. We see how the mitochondria group, again, develops a fractional shortening that equals or improves, slightly improves the ones seen in the DPP group in blue. And again, we see that at every time point during the two hours of loaded profusion, there is statistically significant difference between the mitochondria group and the vehicle group. When analyzing the tissue for infarct size, in both the neonatal and periodic hearts, we find a very significant difference in the infarct size in the vehicle hearts, here, present in black, compared to the mitochondria hearts in red and the DVD hearts in blue. In the graphic showing the degree of edema that the tissue experiments experiences, we can observe how there is no significant difference between the DCD-HARS-3 with vehicle, mitochondria, or the DVD hearts. In the histology analysis on your left, we can appreciate how the hearts treated only with big, with vehicle solution had a lot, a lot larger interfibrular separation compared to the DCD-HARS-3 with mitochondria or the DVD hearts. So with all these results, we can conclude that mitochondria transplantation enhances DCD-HARS-3 in both neonatal and periodic hearts. Preserves myocardial function, equal or better than the DVD hearts. Preserves cellular variability, again, equal or improved convertivity hearts. And it can potentially increase the use of DCD-HARS with mitochondria transplantation, can potentially increase the pool of donor hearts for donation in these groups. I would just thank my department, Dr. McCulley, Dr. Delnido, and all the co-authors of this project. Thank you. Great talk. One of my questions is related to I know at least with livers, when we do something similar with DCDs, the time limit that we tend to set in our heads is about 30 minutes. Part of that reason for that is related to the warm estimate of time that's associated with a critical loss of ATP, considering your transplanting mitochondria. Have you looked at ATP levels and the tissues to see how that changes with the transplantation of the mitochondria? After the mitochondrial transplantation, we look at the ATP levels. With like vehicle and the mitochondria, I would be interested to see how that changes, and I bet it's better with the mitochondrial transplantation. Not in this study, and we've seen how the function on the, but we haven't checked the specifically ATP levels on the myocardial tissue at the end of the experiment. We are waiting on RNA C and proteomics, but not ATP. We haven't looked at that specifically. Sorry. Related to that question, is there, is it possible to sort of basically inactivate the mitochondria before you're being trans injected to make sure that it is really a mitochondrial function that is responsible for the effect that you see? It's going to do inactivate the the activity. Oh, to basically use a poison for mitochondrial activity to make sure that when you transplant mitochondria, there are many other things probably that might be also a cop being. Our previous study in coronary, not by me, but in our lab, like in coronary blood flow, has seen that when you inject just ATP, it has a peak effect, but it's a lot shorter, and it doesn't have the duration of the prolongation. How long it lasts the effect compared to when you inject the mitochondria? That's on our coronary blood flow project. Do you know what's happening? The mechanism of the mitochondria. So for what we know, as of right now, we know that they get included into the cell by a mechanism by its endosomes, and they get into the lysosomes. Part of the mitochondria gets hydrolyzed, and part of them gets fused with the native mitochondria. And we've seen retrograde and anti-grade communication with the nucleus, but the exact mechanism is for sure something that we need to continue studying. Thank you. Any further questions? I'd like to thank all the speakers and for you to come and support the fellows. I think it's very important for faculty to show up and show support for the excellent research that is being done. And I'd like to again congratulate all the four speakers for their very excellent presentations. Now, just as I'm... Lastly, I'd like to thank the Department of Surgery for letting us utilize their grand rounds this morning. As I said, this is the 24th year that we have done this, and I encourage everybody to look at the wiki site and see all the abstracts that have been uploaded, all the posters that have been uploaded based on the abstracts that were submitted. Thank you all and have a great day. Appreciate it.