A--Animal Facility Research and Developement

STATEMENT OF WORK A. GENERAL GUIDANCE 1. Title of Project: Protection of the Brain by Chemical Hypothermia, PI: Shan Ping Yu, MD, PhD 2. Scope of Work: The Contractor shall provide animal facility, pathological, cellular, molecular biological measurements and behavioral/functional tests including MRI imaging for the project entitled. We are requesting 4 option years. 3. Location of Services Provided: Emory University School of Medicine and Emory Yerkes Center 4. Work Hours: The contractor shall be available during normal business hours of 8:00am - 4:30pm Monday - Friday excluding Federal Holidays at a rate of 20 hours per week. Period of Performance Base Year: January 1, 2014 - December 31, 2014 Option Year 1: January 1, 2015 - December 31, 2015 Option Year 2: January 1, 2016 - December 31, 2016 Option Year 3: January 1, 2017 - December 31, 2017 Option Year 4: January 1, 2018 - December 31, 2018 5. Federal Holidays: New years Day, Martin Luther King's Birthday, President's Day, Independence Day, labor Day, Columbus Day, Veteran's Day, Thanksgiving Day, and Christmas Day and any other day specifically declared by the President of the United States to be a national holiday. 6. Type of Contract: This shall be a firm fixed price contract B. GENERAL REQUIREMENTS: Experiments performed will include animal's stroke surgery, measurement of the hypothermic effect using the neurotensin agonists, assessments of pathological, and cellular and molecular biological changes and behavioral changes. Provide animal stroke model and examination of hypothermia therapy and delineate the underlying mechanism of the treatment, including data collection, data analysis and publications. C. SPECIFIC MANDATORY TASKS AND ASSOCIATED DELIVERABLES Animals: Sprague-Dawley rats will be used for the focal stroke models, the use of this species will allow us to compare the result with previous investigations. According to Medline research on 05/02/2011, there is no computer model available for chemical hypothermia treatment in stroke for animals or humans. SD rats are commonly used animal models in stroke investigations, based on the similarity of their brain blood vessel anatomy to that in humans, ready availability, and relatively low cost. SD rats will be used to facilitate comparisons with our previous experiments and to others' data. The power analysis (0.969) shows that 15 animals will usually be included in one experimental group to achieve statistical significance in data analysis. For each test we will usually have 3 groups: sham control, stroke only and stroke plus drug. General veterinary care of the animals: Animals will be kept in animal facility for 2-3 days before surgery and 3 to 28 days after surgery. Veterinary Care for long term survival of rats: The animals will be returned to cages with clean bedding and food and water will be available. They will be observed for signs of dehydration or distress. Postoperative analgesia will be provided every 8 hrs as needed by administering buprenorphine (Buprenex) (0.05 mg/kg s.c.) through 30 gauge needles. The cages will be placed in a temperature controlled room at ~21oC. The light will be switched off at night and noise stimuli will be avoided. Surgery Specifications: All animal surgery will be performed under anesthesia and analgesic drugs/antibiotics will be applied if necessary for post-surgery treatments. For barrel cortex ischemic stroke, rats will be anesthetized using 3% isoflurane in a gas mixture of 70% N2O and 30% O2. Following induction of anesthesia, 1.5% isoflurane will be maintained with mechanical ventilation. Surgical procedures to induce barrel cortex ischemia in adult rats have previously been reported in our published papers (e.g. Wei et al., Brain Res 1022:54, 2004). A 6-mm cranial window will be created using a dental drill and the dura will be kept intact. Using intrinsic optical imaging, the barrel fields will be located and four to six small branches of the MCA serving the barrel cortex will be ligated using a 10-O suture through the dura. Creation of the whisker-barrel cortex ischemia will be completed by bilateral occlusion of the common carotid arteries (CCA) for half an hour followed by reperfusion. In sham controls, ligatures will be placed through the dura and under both arterial branches and the CCA but not tied. Animals will be returned to cages for recovery after regaining consciousness following surgery. For transient and permanent focal ischemic stroke models, rats will be subjected to anesthesia as above. Focal ischemia will be induced following our previously described suture insertion procedures (Wei et al., Neurobiol Des, 19: 183, 2005; Li et al., J Cereb Blood Fl & Met 27: 1043, 2007). Occlusion of the right side MCA will be achieved by advancing a silicone coated occluding thread from the external carotid artery into the lumen of the internal carotid artery to block the origin of the MCA. Reperfusion will be allowed by withdrawal of the filament after 90 min MCA occlusion. The 90 min occlusion time has been widely used in previous investigations, as it induces reliable brain infarction but does not increase mortality. Upon finishing surgery, the surgical wound will be closed using surgical glue and animals will be returned to cages for recovery. In all experiments, animals will be given 2-3 days rest after arrival at our animal facility. They will be subjected to fasting the night before surgery. No water restriction will be applied. Animals will have free access to food and water after recovery from anesthesia. All rats will be kept in air-ventilated cages with room temperature maintained at 22 0.5o C. Veterinary Care for Long-term Survival of rats: The animals will be returned to a cage with clean bedding. They will be observed for signs of dehydration or distress. Postoperative analgesia will be provided: buprenorphine (Buprenex) 0.05 mg/kg s.c. through 30 gauge needles every 8 hrs as needed. The cages will be placed in a temperature controlled room at ~22oC that is on a 12hr/12hr light/dark cycle and noise stimuli will be avoided. The procedures to be used for animal care are consistent with those established by the National Institutes of Health. Every effort will be made to ensure that potential discomfort, distress, and pain will be limited to that which is unavoidable in the conduct of scientifically sound research. Method of euthanasia to be used and the reasons for its selection: At specified times after stroke, or in the event that animals show severe discomfort, infection, or signs of pain, rats will be euthanized with an overdose (100mg/kg) of pentobarbital which is a rapid and potent compound that has been used in previous investigations. This method is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. Equipment Requirements: The equipment must be within walking distance of each other for the safety of the animals involved. Transporting them to different locations for surgery, care, and testing could have very negative results for the animals and for the research. Among other equipment for biological, morphological and cell biology studies, the core facility must be equipped with a StereoInvestigator System (MBF Bioscience, Chicago, IL) operated with an Olympus BX51 fluorescent microscope armed with a motorized stage. This system is ideal for stereological cell counting. There needs to be a second Olympus BX51 microscope with dedicated MCID Elite image analysis and a fluorescence system. An Olympus IX71 inverted equipped with a live cell (time lapse) image analysis system, equipped with a high-speed Hamamatsu digital camera. Also a Zeiss microscope for bright field and fluorescence imaging, a Leica CM1900 cryostat, and a Microm sledge microtome with cooling stage, a Vibratome machine for tissue sectioning, and high-speed centrifuges are needed. A Zeiss LSM 510 confocal microscope and two multi-photon Laser scanning confocal microscopes (Zeiss LSM 510 NLO META systems) need to be in very close proximity to each other and available for use. MRI imaging will need to be performed with a Siemens Magnetom Trio 3T whole body MRI scanner dedicated for research, and a Bruker 7T animal scanner (BioSpec 70/30 USR, Bruker BioSpin MRI GmbH, Ettlingen, Germany) interfaced to a console (SMIS, Surrey, England). The center is specialized in biomedical magnetic resonance at high magnetic fields, focusing on functional brain imaging, high-field imaging, in vivo spectroscopy, and molecular imaging. Training, technical support and dissemination of knowledge related to MRI expertise must be available for every scan, as well as, veterinary support in case of emergency. Data Provided: Provide experimental data in the forms of figures, tables, and manuscripts. The experiments to be performed are required to involve the use of the focal ischemic stroke models targeting to the barrel cortex region. D. REPORTING REQUIREMENTS: The contractor shall deliver the work before the deadlines set by project PI at each stage. Monthly meeting with the PI and co-investigator of the project and quarterly report to summarize the progress of the experiments. E. QUALITY ASSURANCE: PERFORMANCE STANDARDS REQUIRED SERVICE PERFORMANCE STANDARD MONITORING METHOD INCENTIVE/DISINCENTIVES FOR MEETING OR NOT MEETING THE PERFORMANCE STANDARDS Animal stroke model Brain infarct formation, neuronal cell death and behavioral deficits Brain slices, pathological and imminostaining images Co-authorship in published papers, opportunity of attending national/regional meetings for finished specific aims. Additional training and surveillance in the event of slow progress. Pathological and histological measurement of brain sections and cell cutlures Raw data spreadsheets/recordings with statistical analysis; publish quality of image results and quantifications. Supervised by PI and co-investigator on weekly basis. Co-authorship in published papers, opportunity of attending national/regional meetings for finished specific aims. Additional training and surveillance in the event of slow progress. Measurement of hypothermic effect in animal models and comparison between rodents and monkeys Monitoring of physiological parameters Temperature probes, physiological monitors and MRI imaging Co-authorship in published papers, opportunity of attending national/regional meetings for finished specific aims. Additional training and surveillance in the event of slow progress. F. GOVERNMENT RESPONSIBILITIES: Provide the study protocol, directions, and timelines to the contractor for optimal performance. G. CONTRACTOR PERSONNEL SECURITY REQUIREMENTS Meet the security requirements at Emory University and VA medical center. H. INTERFERENCE TO NORMAL FUNCTION: n/a