CBR Research Grants
Project Summaries—2008
2011—2010—2009—2008—2007—2006—2005—2004—2003—2002—2001—2000—1999—1998—1997—1996—1995
Understanding the Impaired Developmental Competence of Ovarian Stimulated In Vitro matured Oocytes Development and Evaluation of a Degradable End-to-End Microvascular Suture Repair of Rat Iliac Artery Effect of Orally Administered Levothyroxine on Hepathic Lipogenesis Topical Adjuvants on Wound Healing in Various Rat Models Testosterone and Cognition: A Non-Human Primate Model DMBT-1 Expression, Atypical Hyperplasia and Gail Model Risk Development and Experimental Studies on Compact Microwave Imaging Array Chamber Demonstrator for Breast Cancer Detection |
Understanding the Impaired Developmental Competence of Ovarian Stimulated In Vitro matured Oocytes Oocytes of mammalian species are ovulated at the metaphase II (MII) stage of meiosis, the only stage at which they are competent to be fertilized and support development of high-quality embryos. Eighty percent of the oocytes collected from ovarian stimulated human in vitro fertilization (IVF) cycles are mature at the MII stage and are therefore ready to be fertilized; the other 20% of oocytes are immature and arrested at the germinal vesicle (GV) stage, which is the stage prior to the initiation of maturation. Importantly, in some patients, the number of immature oocytes retrieved under normal cycles, or even after ovarian stimulation increases in dramatic form. For example, women with Polycystic Ovarian Syndrome (PCOS), which affects about 8-10% of women of reproductive age and represents approximately 10% of the patient population in our clinic, have multiple antral follicles and most of them contain immature GV stage oocytes. Likewise, there are patients who fail to respond to ovarian stimulation treatments and others who for unknown reasons show a high incidence of immature oocytes, even after repeated stimulation cycles. It is clear that the chances of these patients to achieve conception by IVF procedures will be enhanced if reliable oocyte in vitro maturation (IVM) systems were available in these clinics. Here we hypothesize that human oocytes matured in vitro have impaired cytoplasmic maturation. This proposal will focus on evaluating in in vitro matured oocytes key mechanisms of cytoplasmic maturation whose optimization is required for initiation of embryo development. Specifically, we will examine, a) Ca2+ responses induced by physiological agonists in in vivo matured vs. in vitro matured oocytes, b) the distribution and reorganization of the endoplasmic reticulum (ER), the egg¡¦s Ca2+ store, and c) the distribution and mass of inositol 1,4,5-triphosphate receptor (IP3R1), the channel responsible for all the Ca2+ release upon fertilization, which is the signal that induces resumption of meiosis and the initiation of mitotic cleavages. Elucidating the mechanism(s) that underlie the developmental competence of human oocytes will deepen our understanding of mammalian oogenesis and will lead to science-based clinical approaches to the treatment of human infertility. Specific Aims: SA2: To examine the redistribution of the ER in human oocytes. The working hypothesis is that human oocytes undergo atypical redistribution of the ER when maturation is performed under in vitro conditions. This hypothesis will be tested by injecting a fluorescent ER marker, DiIC18, followed by examination of ER organization at different stages of maturation. Freshly collected in vivo matured oocytes will serve as positive controls. Fluorescence will be examined under confocal microscopy. SA3: To determine IP3R1 localization and concentration during maturation. The working hypothesis is that during maturation IP3R1 distribution changes and its concentration increases, although the magnitude of the changes may be diminished by presently used in vitro maturation protocols. We will examine IP3R1 distribution at different stages of maturation and at MII using indirect immunofluorescence (IF) with a specific IP3R1 antibody followed by confocal microscopy. IP3R1 concentrations will be assessed only after completion of maturation, at the MII stage, using Western blotting and receptor concentrations will be compared between in vitro matured vs. in vivo matured oocytes. Consent of Patients: Source of material: Following approval of our protocol by the institution¡¦s IRB, we will seek oocytes from patients as follows: 1) immature oocytes (GVs or MIs) will be collected at the time of follicular aspiration during regular IVF cycles; these oocytes would otherwise be discarded (20% of all IVF oocytes), and 2) 1 mature oocyte (MII) from IVF patients with 10 or more MII stage oocytes, which will serve as the control oocyte. The collected immature oocytes (GV and MI) will be matured in vitro for up to 24 hours. After in vitro maturation, those oocytes that have achieved maturation, as judged by release of the primary polar body, will be evaluated for the aforementioned parameters. Matured oocytes from patients will be used as experimental controls. In vitro maturation will be performed at Baystate IVF. After maturation is assessed de-identified samples, coded with study ID numbers will be shipped anonymously to UMASS Amherst for additional assays. After assessment samples will be discarded no samples will be stored for more than one week. The oocytes will be incubated and monitored for growth then assayed for identifying markers. Data Storage: Patients protected health information (PHI) collected for this study will be information gathered from patient medical records and new information created as a result of this study. Data will be stored on Excel spreadsheets on the computer of the PI Dr. Teru Jellerette which is password protected. Patient identifiers will be removed and data will be transmitted to UMASS in the form of an email directly to the research assistant at the University, Hoi Chang Lee and stored on a lab owned password protected computer. Data will be backed-up on a flash drive and a hard copy will be stored for three years at Baystate Medical. Oocyte maturation The collected immature oocytes (GV and MI) will be matured in vitro up to 24 hours. After in vitro maturation, those oocytes that have achieved maturation, as judged by having reached the MII stage, will be evaluated for the aforementioned parameters. Matured oocytes from donors or patients will be used as experimental controls. If patient withdraws participation: If the patient decides to withdraw their consent samples will be discarded. Effective date: 2008 Development and Evaluation of a Degradable End-to-End Microvascular Suture Repair of Rat Iliac Artery To develop a degradable end-to-end microvascular anastomotic coupler (DEMAC) that will rival or improve the survival of the arterial target when compared to conventional microsurgical techniques. In the field of reconstructive surgery, autologous free tissue transfer is a widely accepted means of providing coverage, volume, and contour for various wounds. For example, women who have undergone mastectomy for breast cancer may elect to have their breasts reconstructed using their own tissue rather than with implants. One such procedure involves removing skin and subcutaneous tissue from the abdomen supplied by a small deep inferior epigastric perforator (DIEP) artery and transferring this free flap to the chest for breast reconstruction. This so-called DIEP flap has many advantages: it avoids the potential risks and complications of having foreign material implanted into the body, it does not sacrifice muscle from the abdominal wall and thus decreases donor site morbidity as well as the possibility of abdominal wall hernias, and also often improves the contour of the abdomen in much the same way as an abdominoplasty does. The procedure entails making an end-to-end anastomosis of the DIEP artery to the internal mammary artery with microsurgical techniques. At times, this procedure is performed in an irradiated surgical field where poor wound healing and infection pose significant risks. The performance of microvascular surgery requires specialized surgical training and is extremely time-intensive. With the introduction of the operating microscope, graft survival has improved due to the increased patency rates of the vascular anastomoses. Even in the most experienced hands, a microsurgical procedure takes a significant amount of time due to actual operating time which includes preparation of the operating microscope for use. This in turn translates into an increased amount of time under general anesthesia for the patient as well as increased operating room costs. In recognition of these facts, there is a demand for finding ways to expedite the vascular anastomosis portion of the procedure. One study found that microsurgery can be performed either under loupe magnification or operative microscopy without much difference in vascular patency rates which saved a significant amount of time1. The next logical step in the evolution of microvascular surgery was the development of numerous vascular anastomotic devices. Some institutions have found that the use of an anastomotic coupler device in breast reconstructions saves significant time and eases some of the difficulty associated with microsurgery. The Unilink is the most well-known and most widely used sutureless coupling system Arterial anastomosis is not a widely accepted use for the Unilink coupler because of concerns for patency due to the thickness of the vessel wall. There have recently been some reports of use of the Unilink in creating arterial anastomoses, however the coupler was suitable A device created by Ethicon included the advantage of being bioabsorbable9. The device was used to reconstitute severed arteries and veins in traumatically injured limbs. The authors found that this device could be used successfully in a hostile environment where an infection could threaten the viability of the target. This coupling system required multiple components and the authors described considerable difficulty applying the device due to certain characteristics of the vessels such as inability to evert the ends and pliability. There is a demand for efficiently creating reliable microvascular anastomoses. We are proposing the development of a microvascular coupler that is reliable, widely applicable, simple to employ, and degradable. We are confident that we can design and create a device using PLA so that operative time is reduced, the anastomosis heals, the device degrades, and the patient is left with a wellperfused arterial target. With the proposed degradable end-to-end microvascular coupler (DEMAC), there would be a minimal learning curve as the coupler is a single unit without moving parts. The placement mechanism proposed is intuitive and only requires that the blood vessels be fit over the DEMAC securely. It is potentially useful in any arterial or venous The technology employed in this project may be used to produce a variety of similar devices for application in a multitude of procedures in several surgical specialties. Applications include use in the creation of hemodialysis access, coronary bypass surgery, limb bypass revascularization procedures, traumatic and oncologic reconstruction, organ transplantation, genitourinary procedures, and more. It would also allow surgeons who are not specifically trained in microsurgical techniques to expand their practice to include some microvascular interventions without the requisite learning curve associated with current anastomotic devices. We expect that with the properly designed and manufactured device, we will maintain or improve the patency rates of the conventional microsurgical iliac repair, and at the same time reduce the amount of time required to complete the procedure. We expect no difference in infection rate between the two groups. Effective date: 2008 Effect of Orally Administered Levothyroxine on Hepathic Lipogenesis Hypothyroid patients on replacement with oral thyroxine frequently gain weight1,2 (T4; to distinguish endogenous T4 from the exogenous T4, we will call this levothyroxine or L-T4). We hypothesize that weight gain is a result of increase stimulation of liver lipogenesis by thyroid hormone. While the mayor product of the thyroid, T4, is constantly infused into the systemic circulation, L-T4 is given as a single oral daily dose and is absorbed by the portal vein reaching the liver in high concentrations. The liver is rich in Type-1 deiodinase (D1) which converts T4 into a >10 times more potent hormone, 3, 3',5'-trioiodothyronine (T3). Thus, this form of administering L-T4 may cause a relative "hepatic thyrotoxicosis" leading, among other effects, to increased lipogenesis, glycolysis and gluconeogenesis and further stimulation of T4 to T3. In rats, the liver is highly sensitive to the lipogenic effect of T3, hence L-T4 may cause increased lipid synthesis and export to other tissues. The aim of this study is to test this hypothesis by assessing liver lipogenesis in euthyroid patients undergoing thyroidectomy, both before surgery i.e. when T4 is normally secreted into the systemic circulation, without exposing the liver to high concentrations of it, and then after surgery, when they are in a steady state treatment with L-T4. We will further test the hypothesis that newly formed fatty acids are exported from the liver in the form of triglycerides (TG), for which we will measure TG production rates in both situations, each patient being his own control. We intend to study 20 individuals, ages 18 to 64, avoiding confounding variables, as detailed in the body of the research protocol. One intriguing observation is that patients with undergoing surgery for thyroid cancer, taking slightly higher doses of L-T4 to suppress TSH, do not gain weight, as do those given merely replacement doses to keep TSH within normal limits. This is an intriguing difference that deserves being investigated. Excess lipogenesis under TSH-suppressing doses of L-T4 may be compensated by oxidizing fatty acids in heart or muscle. Therefore, we will study 10 of these individuals undergoing thyroidectomy for benign conditions, hence on L-T4 doses aimed at normalizing TSH, FT4 & FT3, and 10 individuals undergoing thyroidectomy for thyroid cancer, in whom the L-T4 dose will be titrated to reduce TSH below 0.1 microU/ml. Subjects will be examined after 14-16 hours fasting, when TG liver production is in steady state and TG production rate can be calculated from the fasting concentration of TGs multiplied by their clearance rate. This latter will be measured following the disappearance rate of TGs injected as a 20% Intralipid(TM) fat emulsion injected in a vein, following which we will measure TG concentrations at 5 minute intervals for 30 minutes. Additional measurements will include glycerol, fatty acids, and beta-OH-butyrate to control for rapid lipolysis of injected TGs in Intralipid or the export of lipids from liver being in the form free fatty acids, and for fatty acid oxidation. Since It is known that high carbohydrate (CH2O) supply amplifies the lipogenic effect of thyroid hormone, in the first phase of the study, all patients will be studied after 1 week on a high CH2O diet, to magnify the expected effect of L-T4. In a second phase, we will examine the effects of dietary CH2O. For this, we will examine patients already on L-T4, and will repeat the study comparing in a paired manner the same patient on a low and a high CH2O diet, in a random sequence. Measurements will depend on the findings of the first phase, being limited to TG production rates or another measure of liver lipogenesis, for example, if lipids are exported as FFA instead of TGs. Significance: Results from the proposed study will establish whether TH stimulates liver lipogenesis in humans. They will also show whether L-T4, given as a single oral dose, preferentially affects the liver and whether there is a difference between replacement and suppressive doses of L-T4. This latter comparison may provide insight into potential mechanisms that can be the focus of future research proposals; for example, whether higher doses of L-T4 stimulate preferentially muscle lipoprotein lipase and fat oxidation in heart and muscle. Lastly, these findings may lead to the search for different ways to give L-T4 to avoid the untoward weight gain. Effective date: 2008 Topical Adjuvants on Wound Healing in Various Rat Models The prevalence of chronic medical illnesses rises as the American population ages. The incidence of obesity and diabetes is also increasing among the population. These factors have contributed to an explosion of chronic, non-healing wounds. There is a need for the development of novel therapeutic agents and techniques which will speed healing and can significantly impact the health care system. Skin is the largest organ in the body. It functions as a protective mechanical barrier against environmental insults and serves to retain body heat and fluids. Injuries to skin and the underlying connective tissue represent a major challenge for a variety of physicians and other healthcare workers. Wound healing involves a myriad of complex process and interacting components. Surgeons and pathologists have defined the major cellular components, tissue processes, and several humoral factors involved in normal wound healing. Wound care research has identified several barriers to the wound healing process, including heavy conolization with the bacteria, ischemia, and epithelial cells exhibiting premature contact inhibition. Current concepts in the ideal wound healing process include low bacterial bioburden, adequate nutrients including oxygen and a moist wound environment. Topical adjuvants and wound dressings, along with surgical debridement when necessary, attempt to eliminate the barriers to wound healing and provide an environment to foster healing tissues. The experiments use a rat model of partial thickness wounds which has been well established for the study of wound healing from previous studies. Male Wister rats wil serve as wild-type models. Male Zucker rats have a predisposition to developing hyperglycemia and will serve as our diabetic rat models.Partial thickness wound will be employed first in our pilot study. However, our model has the potential to investigate full thickness (excisional) wounds and even burns. In Aim 1 we will develop a novel rat model whereby each rat will have two identical wounds on it’s back. One wound will be treated as the control condition and the other will be treatedwith experimental dressings. In this fashion, each animal will serve as it’s on control. We expect that the PFOB-alginate dressings will accelerate healing of partial thickness wounds in rats as compared to dressings without PFOB. This effect is expected to be enhanced in diabetic rats as compared to wild –type rats. Wound healing in rats has been shown to be between 10-21 days. We expect that partial thickness wounds in our study will heal within 14 days (our endpoint) with the adjuvants. Effective date: 2008 Testosterone and Cognition: A Non-Human Primate Model Effective date: 2008 DMBT-1 Expression, Atypical Hyperplasia and Gail Model Risk Effective date: 2008 Development and Experimental Studies on Compact Microwave Imaging Array Chamber Demonstrator for Breast Cancer Detection Effective date: 2008 |