Catherine, it's just about 7 o'clock. Do you wait a minute or so? Yeah, we usually wait till 7:01. OK. OK. Give everybody a chance to walk into the room, right, so to speak. OK. OK. Good morning, Steve. Morning, Stu. Yeah, this is a great day. For the hospital. It sure was, yeah. Nothing to, nothing to take for granted. No, nothing to take for granted. So, I think it's 7:01. Catherine, is that OK? Can we get started? OK. So, I wanna welcome everybody, or Doctor Kriedberg and I wanna welcome everybody to uh the uh Judah Folkman Research Day. This is actually the 22nd year that we have, uh, had this event, um. And this year, uh, despite the pandemic, we had, uh, almost a 30% increase in the number of abstracts that were submitted for the presentation today. Um, so, uh, it seems like it's an absolute tradition that this is, um, Gonna keep going and uh this and despite the problems that we had in the past year, um, it looks like it's as strong as ever. So, um, with that in mind, I'm gonna introduce the first speaker, Ken Zhao, um, From, uh, uh, who will talk about a novel orally available uh WNT pathway inhibitor for the treatment of metastatic, uh, cancer. He's from vascular biology. No, Kun, you wanna share your screen? All right, I'm just gonna start. Good morning, everyone. My name is Kunzhou from Bruceetter Lab, Vascular Biology Program. Today, I'm going to introduce a novel approach to block wind signaling pathway in metastatic cancer cells. Cancer metastasis accounts for more than 90% of cancer deaths. And if you look at the 5-year survival rates between localized cancer and distant or metastatic cancers, you will notice this dramatically drop of 5-year survival rates, possibly due to the, due to the acquired new mutations and different tumor marker environment that makes the distant cancer becomes insensitive to, to the therapies that has been shown effectively in localized cancer. So there remains the urgency to find a new treatments against metastatic cancer. In order to do that, our lab started with more than 1000 FDA approved non-cancer drugs and screen them on metastatic cancer cell lines, further identify lead compounds, and eventually determine the mechanisms and make further improvements if ever needed. From our initial drug screen, phenbendazole was a hit. Pnbendazole belongs to a drug family called benzmitazos, which are anti-parasite drugs. We later expand the study to other benz mutilles. As you can see from the table, 5 out of 9 benzammucilles have shown selective toxicity towards two highly metastatic prostate tumor cells, PC3M and PC3MLN4. Pre actually previous literature have already indicated that many benzametazolees such as mebeazole and famenazole have shown the broad anti-cancer effects in multiple types of cancer cells, which supports our finding here. However, before we translate this finding into clinical studies, there are two major problems for Benz measles. The first problem is it has a poor water solubility. And because of the poor water solubility, it has a poor in vivo bioavailability. So, to improve that, we collaborated with Doctor Li Jun Sen's lab from Beth Israel, who retained the active moiety of mebenazole. While adding the oxygene groups to increase the solubility. And then finally came up with our lead compound OBD-9. Later in the solubility test, we have seen a significant Increase of the solubility in OBD9 compared to mebenazole. So now we have a lead compound OD 9 with improved solubility. What about the cytotoxicity? To test that, we actually performed the cell viability assay in multiple types of cancer cell lines, including lung cancer, prostate, and ovarian. As you can see from the table, both OD 9 and mebenazole have shown similar EC50 values, which indicates that OD 9 retains cytotoxicity of mebenazole in vitro. So, what about in vivo? To test that, we have used the A54 line lung cancer subcu xenographs, where we orally treat with either mebedazole or two different dose of opening 9. At the end of study, when we compare the treatment volume, While we did not see a significance between mebenazole and we group, we have seen a significant reduction of tumor volume between OD 9 and mebenazole, both at 30 mg per kilogram. So this study indicates that oral OD 9 significantly inhibits tumor growth in vivo. So now the question is, what is the maximum behind the opening night? In order to find the best cell model to study that, we actually performed the NCI 60 drug screen, basically, is to test the opening 9 in 60 different cancer cell lines. Although the data are not showing here, we have found that OD 9 works best in colorectal carcinoma cells, which is CRC. CRC is the 3rd leading cause of cancer-related deaths in the US and about 25% of patients present with metastatic disease. While treatment options are limited for metastatic CRC, majority of them are chemotherapy. And more than, and one important fact about CRC is that more than 90% of CRC exhibits hyperactivated wind signaling. So before we move the study of opening 9 in CRC, we're gonna first to confirm the anti-cancer effects of opening 9 in colon cancer cells. So, we have used the two different colon cancer cell lines, including these metastatic AWP 620 cells. In this cell viability essay, we have noticed the significant reduction of the cell viability as low as one micromotor compared to vehicle group. So these results suggest that OD 9 inhibits colon cancer cell viability. So now the question is, what is the potential maximum of action for OBD9 in colon cancer? Previous in silicon modeling predicts that mebendazole interacted with the TRAV-2 NCK interacting kindness clinic. And published reports also suggest that tinic remains to be an important player in wind signaling pathway in colon cancer. If you look at this canonical windscreening pathway, clinic is majority localized in nucleus by phosphorating TCL4, thus activating the betaclein TCL4 transcription complex, and then regulate several downstreaming substrates including LRP5, CEMI, and TCL4 itself. Because OBD-9 is a derivative of mebendazole, our hypothesis is that clinic is a potential drug target of OBD9. In order to test this hypothesis, we first ask the question, does OD9 affect the kindness activity of clinic? So, we have performed the self-free kinase activity essay by incubating the recombinant tinic protein with recombinant TCL4 protein in the presence of different dose of OBD9 or NCB 846, which is a positive control as a tinin kinase inhibitor. So, unlike NCV 846, where we have seen a significant reduction of the phosphorcene levels, even at the highest dose 150 micromolar opening night, we still cannot see the significant reduction of the phosphorcene levels compared to control. So these results indicates that opening line does not inhibit the kindness activity of clinic. So, if it's not the kind of activity, what about the messen DNA level or protein level? So to study that, we treat the colon cancer cells with different dose of opening 9 for 4 hours, and then either perform QPCR to detect the tin messen DNA level, or Western blood to to detect the tinin protein levels. While we did not see any significant difference between OBD 9 and weak groups, we have seen a significant reduction of the clinic protein levels after OBD-9 treatments. So these results suggest that over downright glythenic protein level, not messing on the level. We have further studied if if OD9 treatment will cause any functional consequence of the wind signaling. So, we have treated the cells with different dose of OD99 and perform the QPCR to detect several transcripts of the wind downstream targets, including TCA4, ARP5, AXN2, and SEMI, where you can see the significant reduction of the transcript levels. For in vivo study, we have used the SC 116 xenographs and the orally treated with two different dose of OD 9. At the end of the study, when we compare the tumor volume, we have observed a dose-dependent tumor, tumor reduction in OE 9 treatment groups compared to vehicle control. These results indicates that Or OD9 significantly inhibits colon tumor growth. And later, in the IG staining in the tumor tissues collected from the in vivo studies, we have confirmed the reduction of the tinic staining and KI-67 staining in the opening night treatment groups. So to summarize here, we have identified an orally available drug potentially against the metastatic cancer. And we have shown that Oberlin inhibits colon cancer cell growth and down regulates protein levels of tinic, and it can also down regulate the windscreening pathway. For future directions, we plan to identify the detailed working maximums of opening line down regulating genic protein levels. Is it via translational control or post-translation control? We also to study OD9 in other metastatic colon cancer cell models as recently obtained a circulating tumor cells derived from colon cancer patients. Last but not least, is to investigate Olinn in combination of treatments such as chemotherapy or cancer immunotherapy. With that, I'd like to thank the, uh the uh organizer for giving me the opportunity to present my work. I want to thank the support from my PI Bruce Eter, and also all other collaborators, and most importantly, the Vascular Biology Program. Thank you. Thank you, cun. Um, Are there questions, uh, For him. Uh. It's hard to see. One question, let's see who. This is Jordan Creedberg. Could I ask a question? Sure, sure. Yeah. Uh, is, I noticed you use 30 mg per kilogram. Have you ti traded that down to see if lower doses would work? And, uh, is it realistic that a dose of that level could be used in humans, or what are your thoughts about that? Uh, Actually, we haven't, uh, tested the lower dose of OD-9, and that's kind of sort of our next plan is to, uh, decrease the dose of OB-9 to see what's the minimum dose of OB-9 that needed to show the anti-cancer effect. And actually, so besides that, we have also have done some PK studies, and also the, uh, and also the ADMS studies to, to basically, uh, to further understand the characteristics of OB-9. And so far the opening line shows a relatively, I would say favorable profile of the PK and, and of course, further, further studies need, uh, further studies need to be done to, to, uh, further answer your questions. Thank you. Great talk. Hi, uh, may I have a question? This is, uh, Xi from Kirby Center. Yeah, hi. Uh, very interesting compound. Uh, my question is, uh, in Uh, current, uh, many trials, phase one trials of wind-related inhibitory compound, uh, a commonly observed side effect is, uh, bone toxicity, uh, affecting, uh, bone density. Do you know in your mouse model, whether this compound has any effect on, on the bone? Uh, We, so. For, so, so far, for all the in vivo studies that I have performed, I did not notice any significant safety issues and I did not specifically check the bone, bones of the mice. But if I remember correct, to me, they all look relatively fine, like with their bone. I mean, of course, uh, like further studying need to be performed to specifically study whether this opening night would, like, whether opening night treatment will, will, we can't affect their bone density. Yeah, OK, thanks. OK, well, Kun, thank you very much, um. Uh, the next speaker, uh, is, um, Ryan Sanborn, who will talk about uh bridge enhanced ACL repair has higher psychosocial psychological readiness compared to autographal ACL reconstruction at 6 months, uh, following, um, surgery. Thank you for the introduction. Um, hello everybody. My name is Ryan Sanborn. I'm a clinical research coordinator on the BARR trials, which stands for Bridge Enhanced ACL repair. My talk today will initially touch on the background behind the development of the Bayer technology, introduce the clinical trial itself, and then I will focus more specifically on psychological readiness in the BAR 2 trial. Here are our disclosures. As a refresher for the anatomy of the knee, ligaments connect bone to bone. The ACL specifically connects the femur to the tibia, goes through the center of the knee, and keeps the knee stable when you are changing direction. In comparison, the MCL also connects the femur to the tibia but is on the medial side of the knee. ACL injuries affect upwards of 200,000 patients each year. It typically affects young individuals who are especially active. The peak age of injury in women is from 15 to 19 years old. In men, it's between 17 and 25 years old. Regarding treatment, the MCL can heal with a brace, but the ACL requires surgical intervention. Up until the 1980s, ACL injuries were treated by suturing the two torn ends together. However, this method had an unacceptable re-injury rate, and the procedure was widely abandoned. As such, the ACL reconstruction has become the gold standard method for surgical treatment. A reconstruction removes the old torn ACL and replaces it with a tendon graft, which typically includes a hamstring tendon or patella tendon. The question remained, however, why wasn't the ACL healing reliably with suture repair. One theory the team put forward was that inflection, the torn ends of the ACL are nicely opposed to each other, but in extension, a gap forms as seen here. In ligaments such as the MCL, which again sits outside the knee joint, after injury, this gap is nicely filled with a stabilized blood clot as seen here. However, this bridging clot is absent in a torn ACL. The ACL sits in a synovial fluid environment which prevents clot formation. Therefore, without a stable clot bridging the gap between the two torn ends, the ACL fails to heal and ultimately the suture repair fails. As such, we hypothesize that if we could create something to fill and bridge this gap to assist in stabilizing the clot, we could ultimately heal the ACL. The team worked with the FDA to conduct a series of scaffold development studies. Per their requirements, the team manufactured a collagen-based scaffold which was able to hold 5 times its weight in blood, stabilize a blood clot within the knee joint, activate the first part of wound healing via platelet activation, and it was designed to absorb 8 weeks after implantation. Utilizing the scaffold, in vivo testing in pig models demonstrated that ligament histology was restored in the newly grown ligament after bear. The strength of the new ligament was superior to suture repair alone. And equivalent to a graft after ACL reconstruction. Additionally, the procedure preserved the cartilage surfaces. Both untreated ACL injuries and ACL reconstruction demonstrate arthritic changes, which are the two pictures on the left. This is not seen after Bear, the picture far on the right. Based on these pre-clinical results, we obtained FDA approval for a small safety study and then moved on to a randomized controlled trial. We really wanted to look at adolescents as this is who we treat at children's. These patients have highest rate of graft failure, up to 20% by 2 years post-surgery. These patients are also at a very high risk for developing post-traumatic osteoarthritis in their lifetime. If a patient has an ACL reconstruction at 14 years old, there's a high risk for arthritis by their early 30s. The BR 2 trial will be the focus on the rest of this presentation. Doctor Yen was the PI of the study. It was a randomized control trial. 65 patients were randomized to BR. 35 patients randomized to ACL reconstruction. The primary results at 2 years included comparable patient reported outcomes, comparable knee stability, improved muscle strength, and re-injury rates similar to that in ACL reconstruction. And just to go back a little, my apologies. Doctor Yen, Doctor Mikaley, and Doctor Kramer performed all surgeries within this trial. These outcomes led us to ask, how does bear affect psychological readiness to return to sport? For athletes, return to pre-injury level of activity is widely considered a primary outcome. However, up to 2/3 of patients are unable to return to sport by 1 year, regardless of satisfactory functional outcomes. This discrepancy led to an investigation of psychological factors. Psychological factors that were commonly reported and associated with an athlete's unwillingness to return to sport include lack of confidence in the knee, fear of re-injury, and loss of self-esteem. Furthermore, in 2008, a patient reported outcome called the ACL Return to sport after Injury Scale, also known as ACLRSI, was developed to measure psychological readiness to return to sport, and this was specific to ACL patients. The primary purpose of this secondary analysis was to determine if psychological readiness is higher for patients who received the bare procedure versus ACL reconstruction. We also had a secondary aim. To be clear, for this aim, we didn't stratify by ACL procedure and instead looked at the entire cohort, which will be important to note for later results. We believe baseline factors and six-month outcomes would predict higher psychological readiness following an ACL procedure. The ACLRSI scale was used to measure psychological readiness. It was measured at 6 months, 12 months, and 24 months. As seen in the questionnaire, which is on the right, it includes 3 subcomponents, which looks at patients' emotions, confidence in performance, and risk appraisal. For the secondary aim, we honed in on the 6-month ACL RSI score, as this was when surgeons first considered return to sport. And 6-month scores have been used to predict return to sport by 1 year. Data collected in the secondary analysis included demographics and functional outcomes at 6 months. Functional outcomes included knee stability, strength and hop testing, and patient reported outcomes. The IKDC is a common patient reported outcome to to assess subjective knee function. In BR 2, patients were similar in age, sex, preoperative activity, and intraoperative findings between the two groups. Our main finding was that their patients had significantly higher ACLRSI scores at 6 months compared to ACL reconstruction patients, as seen on this graph. Scores were similar by 12 months, and continued to be similar at 24 months. Now getting into the secondary aim of looking at predictors of psychological readiness, again reiterating that we didn't stratify by procedure. For baseline factors specifically that predicted six-month psychological readiness scores, we found that age, contact injury, sports level, and type of procedure were all associated with six-month psychological readiness scores. Stepwise regression analysis showed that treatment and sports level were independently predictive of better six-month ACL RSI scores, explaining 15% of the variability. Patients who pre-injury were participating in level one sports, which include jumping, pivoting, and hard cutting. Such as soccer, football, and lacrosse, were more psychologically ready to return to sport at 6 months, as evident by a 13.3% improvement in six-month ACLRSI score. Additionally, patients who underwent the BRE procedure were also more psychologically ready to return to sport at 6 months, as evident by a 13 point improvement in 6-month ACL RSI score. Bivariate analysis between 6-month outcomes and 6-month ACL RSI scores found that better psychological readiness was associated with better IKDC scores at 6 months, lower pain levels, better strength measures, and longer distances for a single leg hawk. For the final model looking at predictors of six-month psychological readiness scores, the single best predictor was a subjective IKDC at 6 months. This explained 45% of the variability in 6-month psychological readiness score. A 10 point improvement in IKDC yielded a 12.4% increase in ACLRSI. We found that all baseline factors, including the bear's procedure, no longer contributed as a final predictor. We also conducted the multivariate analysis with excluding the IKDC as the IKDC is also a self-reported measure. Increased 6-month ACL RSI scores were associated with lower pain, increased hamstring and quadriceps strength, and less knee laxity at 6 months. Interestingly, although psychological readiness scores weren't used as clearance cri criteria, patients cleared earlier for return to sport had better 6-month psychological readiness scores. In conclusion, our main finding showed that patients who underwent the BR procedure had higher psychological readiness scores at 6 months after surgery. We believe this to be the case because BA doesn't require graft harvest, as does ACL reconstruction, and graft harvest adds morbidity to the surgical procedure. Multivariate analysis of baseline and 6-month outcomes determined that better psychological readiness scores at 6 months were most strongly related to improved IKDC scores at 6 months. Furthermore, when excluding IKDC from analysis, we determined that lower pain levels, better muscle recovery, and less knee laxity at 6 months post-surgery were also associated with improved psychological readiness scores. The bare procedure wasn't included as a final predictor, and we believe this to be the case because of how closely it relates to IKDC scores, hamstring strength, and pain levels at 6 months. I'd like to acknowledge the clinical team and surgeons who made this all possible, uh, with a special thanks to Doctor Christina, Doctor Murray, and Gary for being great mentors on the secondary analysis. Thank you. OK. Uh, thank you very much, Ryan. Are there any questions for, uh, Ryan? Um, OK, maybe I will start with Ryan, could you tell us where you think you'll be going from, uh, here with this based on the results of this study? Well, so this is the bear 2 trial, and we've actually had a 3rd trial, um, Uh, which we are paused to enrollment now. Um, and we actually received FDA approval back in December of 2020, which was a big, big thing for us. Um, and with these results, um, we can potentially use psychological readiness scores to Uh, see when patients are best to go back to sport, um, returning to their sport. Um, so maybe we can add that into clearance criteria along with strength measures and, uh, patients subjective knee function. Great. Thank you. Uh, I'm not seeing any other questions. Is anyone, I have, I have a question. Yeah, go ahead. Uh, this is Sunit Awal. Uh, Ryan, that was a great talk, very clear. Um, I have a, uh, was, were all the patients in the control group, um, hamstring, uh, tendon grafts, or were there some cadaver? And how would that factor into the results in terms of pain and function? Yeah, that's a great question. Thank you. Um, so that's actually one of the limitations to the study, so. Um, this is comparing bear versus, we say autographed ACL reconstruction. Um, so 33 of the 35 patients in the ACL reconstruction group had a hamstring graft. So it may not be generalizable to a patella tendon. Um, and we also, there were no cadaver grafts used in this, uh, study. OK. Thank you very much. I think we will move on. Uh, our next, uh, speaker is Scott Flegor from Vascular Biology who will, uh, tell us about FG 4592, uh, in, uh, bronchopulmonary dysplasia. Uh, Scott. Good morning. Thank you to the organizing committee for the privilege of presenting our work today, investigating FG 4592, a prolyl hydroxylase inhibitor, and improving long-term pulmonary functional outcomes in a murine model of bronchopulmonary dysplasia. I have no disclosures, but a patent has been filed by Boston Children's Hospital on behalf of Doctor Pewter for use of FG 4592 in hypoplastic lung diseases. Bronchopulmonary dysplasia, or BPD is a clinical syndrome of disrupted lung development seen in premature infants. Prematurity, mechanical ventilation, hyperoxic toxicity, and perinatal sepsis, in addition to other factors, lead to lung injury and pro-inflammatory cascades, ultimately disrupting lung development. There is no effective treatment for BPD. Current interventions such as glucochoroids administered perinatally, minimizing barotrauma, and minimizing oxygen toxicity are aimed at preventing lung injury, but do not treat BPD once it is established. Short-term complications include early mortality in the most severe cases, prolonged need for respiratory support, pulmonary hypertension, and ultimately prolonged hospitalization. Many babies require supplemental oxygen when they go home for a period of time. Long-term complications include frequent re-hospitalizations early in life, as well as decreased pulmonary function that persists to adulthood. BPD results from disrupted lung development. Gas exchange depends upon the process of alveolarization or the formation of new alveoli, which normally occurs from 36 weeks gestation up through adulthood. This process is paralleled and dependent upon angiogenesis, or the formation of new blood vessels. On histology, the lung injury is seen as complex and heterogeneous. It includes decreased alveolarization, cystic and emphysematous changes, fibrosis and airway injury. Disrupted alveolarization results in disstensible large alveoli with decreased septa and area for gas exchange. Angiogenesis and microvascular development are critical for alveolarization. Low levels of pulmonary vascular endothelial growth factor, or VAGF are associated with development of BPD. Here, human lung sections are stained for VEGF and brown. BPD lungs have markedly decreased VAGF expression. Pigment epithelium derived factor, or PEDF is an antiangiogenic factor. In a murine model of hyperoxic lung injury, PEDF knockouts were protected from alveolar simplification and the BPD phenotype seen in these slides. Together, these findings suggest that altering the pro and anti-angiogenic environment in the lungs may provide a potential therapeutic modality that accelerates lung development. The hypoxia inducible factor pathway is critical to oxygen sensing and angiogenesis. FG 4592, or oxatostat, mimics hypoxic conditions and inhibits degradation of HIF1 alpha, which is stabilized and translocates to the nucleus, up-regulating proangiogenic factors and down regulating anti-angiogenic factors. We first sought to evaluate long-term structural and functional outcomes in a murine model of BPD and subsequently evaluate whether Roxxatisat improves long-term structural and functional outcomes in the murine BPD model. Neonatal litter mates were randomized to either room air or 75% oxygen for 10 days, after which pulmonary function testing was performed. To perform pulmonary function tests, mice are anesthetized, a tracheostomy is performed, and mice are connected to a ventilator that can measure lung mechanics. Following this, lungs are harvested for lung morphometrics. On pulmonary function tests, hyperroxia led to increased compliance and inspiratory capacity, consistent with an obstructive BPD phenotype with larger, more distensible alveoli. Representative lung histology shows that hyperroxia, seen on right, results in gross alveolar simplification with larger alveoli and fewer septa. This is reflected in lung morphometrics. Mean linear intercept is a measure of average distance between alveoli. A larger distance is consistent with fewer septa and larger alveoli. Hyperoxic lung injury increased the mean linear intercept and significantly decreased alveoli per high-powered field, consistent with alveolar simplification and disrupted lung development. After establishing short-term structural and functional outcomes, long-term pulmonary outcomes are assessed in this model as BPD is a chronic disease with long-term consequences. Neonatal litter mates were randomized to either room air or 75% oxygen for 10 days, after which they recovered in normoxia. At 5 weeks, treadmill exercise tolerance testing was performed. This was repeated at 8 weeks, along with pulmonary function tests and lung morphometrics. In the exercise tolerance test, mice are acclimated to an inclined treadmill with a shock grid at the bottom that delivers a low voltage shock. The treadmill accelerates gradually. Mice are removed at the point of exhaustion, which is defined as staying on the shock grid for more than 5 seconds. At 5 weeks, mice exposed to hyperroxia ran for less distance and time, consistent with decreased exercise tolerance. Decreased exercise tolerance was again noted in the hyperroxia exposed mice at 8 weeks, with decreased distance and time run compared to the Normoxia controls. On pulmonary function tests, the obstructive BPD phenotype persisted at 8 weeks with increased compliance and inspiratory capacity in the hyperroxia exposed mice. This was confirmed histologically. Histology again demonstrated gross alveolar simplification, with fewer larger alveoli and decreased septations. Lung morphometrics demonstrate that hyperoxxy exposure resulted in increased mean linear intercept, the average distance between alveoli, and decreased alveolar count in the hyperoxy exposed mice at 8 weeks. These findings demonstrated that both structural and functional pulmonary outcomes of hyperoxic lung injury persisted to 8 weeks, confirming this is a good model to study bronchopulmonary dysplasia. After establishing long-term outcomes in this model, we sought to evaluate whether Roxatostat FG 4592 improved long-term structural and functional outcomes in this model. Following 10 days of hyperoxic lung injury, or normoxia control, mice were randomized to every other day oral gavage of either Roxxatostat or vehicle control. At 5 weeks, mice underwent treadmill testing, pulmonary function tests, and lung harvest for morphometrics. On exercise tolerance testing, treatment with Roxatastat improved exercise tolerance with increased time run in the hyperroxia exposed mice compared to hyperroxia vehicle controls. On pulmonary function testing, hyperroxia increased compliance and inspiratory capacity in the vehicle control mice. Roxatostat treatment improved compliance and inspiratory capacity in the hyperroxia exposed mice compared to their vehicle control litter mates. Gross lung histology demonstrates that the hyperroy of vehicle control mice demonstrate alveolar gross simplification with fewer large alveoli. Lung sections for hyperroxia exposed mice treated with Roxatace show relatively more alveoli in septations. This is reflected on lung morphometrics. Hyperoxxy exposed controls demonstrated elevated mean linear intercept and decreased alveolar count. Compared to the hyperoxxia vehicle controls, hyperoxy exposed mice treated with Roxatostat demonstrate decreased mean linear intercept and increased alveolar count, which is consistent with accelerated alveolarization. In conclusion, BPD is a common disease of prematurity and disrupted lung development with significant short and long-term consequences. Our model of bronchopulmonary dysplasia demonstrated short and long-term structural and functional effects consistent with the BPD phenotype seen in humans. Treatment of hyperroxy exposed mice in this model with Roxatostat demonstrated improved lung morphometrics, pulmonary function tests, and exercise tolerance. There is currently no treatment for established bronchopulmonary dysplasia. Roxaat has successfully undergone phase 3 trials for treatment of anemia of chronic kidney disease in adults, and a new drug application is under review at the FDA. In future studies, we seek to translate our findings to large animal models of BPD to assess the safety and efficacy of Roxatostat. After establishing safety and efficacy, these findings may be expeditiously translated to the neonatal population. Special thanks to the vascular biology Program in the Department of Surgery for supporting our work, as well as Jenny Yu and the rest of the Pewter lab. I would be happy to take any questions at this time. Thank you very much. Great. Thank you very much, Scott. Are there any questions for Scott? Hey, Scott, uh, quick question for you, um, with regards to side effects for the drug. So prohydroxylases, as you know, are not just important for regulating, um, hip stability, but they also are important in, uh, collagen synthesis. Is there any, any indication that, that this drug affects, um, uh, the prolhydroxylases involved in extracellular matrix deposition or, or, or other things, any indications of Of changes in uh in uh collagen or other, other matrix proteins. That's an excellent question. It's something that we will have to look at. We have not specifically studied that. OK. Are there any other questions for Scott? Jordan, Diane uh Belenberg had a question. Diane, please go ahead. Hi, yes, um, I just had a question if you tested the number of type 1 to type 2, pneumocytes within the alveoli when, uh, between the groups. We have not, and that's part of what we are currently working on. Uh, we are also looking, trying to establish, uh, using immunofluorescence, uh, the actual degree of distal airway angiogenesis, and actually looking at capillaries as well to try and better characterize that, but we have not looked at that. Thanks. Good job. Mhm. OK. Any other questions? I'm not seeing any. OK. Great. We will move on to our, uh, final, uh, speaker who is, uh, Mikayla Benedict from anesthesiology who will tell us about an MRI study of, uh, the impact of morphine administration on rat brain volumes. Mika. Hello, everyone, um, and thank you all for listening. My name is Mikayla Benedict, and I'm a rising senior at Harvard College. The talk I'm giving today will also be featured at the International Association for the Study of Pain's World Congress on Pain. I'll be sharing my research on the immediate and long-term impact of prolonged morphine administration on rat brain volumes through our MRI study. This work was completed under the mentorship of my principal investigator, Doctor Jessica Bajk in the anesthesiology department at Boston Children's Hospital. The goal of our study was to understand the impact of prolonged morphine administration on rat brain volumes in a rat pup model using MRI. We use a reverse translational model to study a phenomenon observed in humans and infant rats. We hope that our data will eventually inform human treatment. We use structural brain MRI analysis in a rat model to a prolonged morphine administration to answer a few questions. First, we studied the immediate effects of prolonged morphine administration in an infant rat model. We then studied the long-term effects of morphine administration in infancy in adult rats. This was divided into two separate analyses. One of the long-term effects of morphine dosing in infancy in adulthood, and one of the effects of subsequently dosing with morphine in adulthood following morphine treatment in infancy. Our goal was to complete A total and regional volumetric brain analysis, which we accomplished through manual brain segmentation. Infant and adult rats received light anesthesia during MRI scanning at the small animal imaging lab at Enders facility. Both infant and adult rats were anesthetized with 3% isoflurane at 1 L per minute for up to 3 minutes prior to being taken into the MRI scanner. Once in the scanner, animals were placed in an animal cradle in the prone position and reconnected to anesthesia delivery system through a nasal cone at a lower anesthesia level to maintain a consistent respiratory rate in the narrow range between 45 and 50 breaths per minute. To the right we have illustrated the isofluorine levels and respiratory rate. We use the ITK Snap software to manually segment brain regions of interest in these Two weighted MRI images. You can see these regions displayed in the coronal, sagittal, and axial sections, as well as a 3D rendering to the right. The regions analyzed with the cortex shown in red, hippocampus and teal, deep gray matter in yellow, cerebellum in green, and brain stem in purple. The brain stem consists of the medulla, pons, and midbrain. We chose to manually segment brain regions due to an absence of available automated tools for infant rat brains. Low image contrast as a result of poor myelination in the infant rat brain poses a challenge for segmentation protocols that likely contributes to the inaccuracy of automatic segmentations. The Paxinos and Watson rat Atlas was used to train a single researcher in segmentation protocol. Segmentations were completed blind to treatment group, and completed segmentations were then verified by a senior researcher. Moving on to our results, we'll start by discussing our infant rat experiments. Our question here was what are the immediate effects of prolonged morphine administration in an infant rat? Twice daily morphine or saline injections were administered over a period of 2 weeks from postnatal day 1 to postnatal day 14. After the treatment period, T2 weighted MRI images were collected of the brain. We want to note here that we did not decide to split our infant groups by sex, because an analysis of total body weight showed no difference between adult male and infant male and female rats. Additionally, the literature does not support sex differences in infant rats. In our first experiment, we found that morphine-treated rat pups had globally smaller brain volumes compared to controls. To the left you can see a graph of mean absolute values of total brain volume and absolute volume of the brain regions listed before, including the forebrain, cortex, deep gray matter, hippocampus, cerebellum, and brain stem. The left graph shows us that morphine-treated infant rat pups had smaller brain volumes across all regions studied. To the right is a graph of normalized brain volumes of each region as a percent of total brain volume. A lack of difference in normalized brain volumes between groups indicates that there were no regional vulnerabilities observed. For our adult experiments, we sought to answer two questions. First, what are the long-term effects of prolonged morphine administration in infant rats? And secondly, what is the impact of prolonged morphine administration in adulthood in the setting of previous administration in infancy? This slide describes the experimental approach we took in this study. Twice daily morphine or saline injections were administered in infancy. Following treatment in infancy, rats underwent a 10-day weaning period and were allowed to grow into adulthood. The rats were treated again with 2 weeks of either morphine or saline treatment, yielding 4 treatment groups. Two weighted MRI images were collected from each group following the 2-week treatment period. In adult rats, we observed sex differences in body weight that were in support of us splitting our groups. Specifically, female rats had significantly smaller body weights than male rats across all groups. Based on these findings, we decided to split our adult analysis by sex. When we raised infant rats who underwent either morphine or saline treatment in infancy into adulthood and then treated again with saline, we found that there were no long-term effects of prolonged morphine administration on total and regional brain volumes in adulthood. In both female rats shown to the left and adult male rats shown to the right, we see no differences in brain volumes between previously saline-treated and morphine-treated groups. This indicates that there are no long-term effects of prolonged morphine use in infancy on adult brain volumes. In our last experiment, we found that prolonged morphine administration in infancy and subsequently in adulthood led to selectively smaller regional brain volumes that differed with sex. Here we compare a twice saline treated control group. With the group treated with morphine only in adulthood, and a group treated with morphine in both infancy and adulthood. In female rats, We found smaller forebrain, cortex, and total brain volumes, specifically in the twice morphine treated group. In adult male rats, we found smaller cerebellum volumes again in the twice morphine treated group. This may indicate regional vulnerabilities upon subsequent morphine dosing that differ up by sex. This leads us to a few conclusions. Firstly, that the mechanisms that contribute to globally smaller brain volumes in infancy following prolonged morphine administration should be investigated. Secondly, the neuroplasticity that allows for full recovery of brain growth remains unknown. And third, that the prolonged morphine administration in adulthood after previous exposure in infancy may lead to brain structure adaptation or maladaptation that differs with sex. Thank you for listening to my talk and I'd be happy to take any questions now. Thank you very much. Are there questions for uh Mikayla? Uh, let me start with, uh, one. did you, uh, uh, uh, it looks like you didn't mention any behavioral tests. Did you even just, uh, looking at their activity in the cage, uh, get the sense that there are any differences in activity or behavior based on treatment or not? Um, that's very interesting. I do have a slide prepared to answer that exact question. Um, so we actually have not yet, um, completed a behavioral analysis on the twice morphine treated group. However, a previous study by my lab did test, um, adult rats treated with morphine in infancy, and they found no difference in, um, behavioral measures across many different studies. Um, we did see a slight Um, decrease in thermal threshold and a slight difference in one hour testing for novel object, um, memory test, um, but other than that, um, across many other measures, there were no behavioral differences when, um, morphine-treated infants were raised into adulthood. Thank you. Other questions, uh, Um, I have a question, Michaela. Did you look at the, um, waiting a period of time in the infant rats and then re-expose them to morphine? Cause, you know, in real life, some patients, uh, some children will have to have more than one operation over time. I think that would be a really interesting follow-up study. That's not something that we did, um, but there are definitely a lot of different directions that we could take, um, this study in the future. Right. Any other questions? OK. If I'm missing any, just please shout out. Uh, let me ask one more then. Were there differences at all in body weight when you, you, it seemed like when you normalized to brain volume, some of the differences became not, not as different. How about when you, did you normalize to body volume? Or rather, yeah, we're actually, um, we haven't completed those statistical analysis yet, um, but we did see differences in body weight, specifically smaller, um, body weight in infants treated with morphine. Um, we would attribute that probably to, um, the fact that we use a split litter or within litter design, um, so morphine treated, um, infant rats slept more. than saline-treated infant rats, so they had a nursing, um, disadvantage. Um, so one direction that we might take this study in the future is to repeat, uh, with a non-split litter design, so we have litters that have specifically morphine-treated and specifically saline-treated pups to, um, prevent that, um, discrepancy. Great, thank you. Bye. Don't think I see any other questions at this time. Mhm. OK, well, if I could just make a comment, uh, Jordan, um, yes, uh, all of those, um, presentations were spectacular and professional, but I think we have to call on and recognize, um, this environment is a pretty senior investigator environment and to have an undergraduate college student make a presentation of that quality, uh, it is, uh, remarkable, uh, fair poised and, and beautifully presented. Uh, congratulations to you. Right. Thank you, uh, point well taken. Uh, I'd like to, uh, thank all the speakers and all the participants for joining us. I'd also like to, uh, acknowledge, uh, the, uh, support and organizing Research day of, uh, Luanne uh, Posey, uh, from the Office of Fellowship Training and Stephanie Schneider, uh, in research computing. I'd like to invite everyone to also, uh, attend medical grand rounds at noon. Where we will have 4 more presentations and then an afternoon session from 4 to 5 p.m. where again there will be uh 4 more presentations. All these uh oral presentations were based on a scoring from a panel of judges and we'd like to also acknowledge the work uh and time that they put into uh supporting Research Day. And finally, uh, there will be a virtual poster session for the next 3 days, uh, where, uh, you know, over 100 posters, uh, will be, uh, available for, uh, you know, taking a look at, and we hope that you will all, uh, take some time to look at the, uh, posters during our virtual, uh, poster session. And of course, we all hope that we will by next year return to an in-person, uh, research day. Uh, so, uh, thank you all, uh, for your attendance, and, uh, that will conclude our session. Thank you.
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