Application for Approval for the Use of Vertebrate Animals in Research and Educational Activities (version )

Transcription

1 Stony Brook University Institutional Animal Care and Use Committee (IACUC) Application for Approval for the Use of Vertebrate Animals in Research and Educational Activities (version ) NOTE: Review IRBNet instructions at as there are other items you MUST upload into this project (registration form, grant(s), housing form, etc). Instructions are first item in Forms/Templates section in IRBNet (left side navigation bar after you log onto your project). ********In addition to this application you must upload the ON-LINE registration form and grant(s): Log onto your project. Your project needs to be unlocked. If locked, call to unlock. Once unlocked, the registration form can be accessed by: o Select Designer on the left hand navigation bar o Scroll down slightly to see "Add New Document" in the box in center of your screen - select this o In the 2nd blue box (On-line Document) and you will be able to access the Registration form for IBC, IACUC & SCRO from the drop down box. Click on Add. o Read each screens carefully as they come up and click next o Fill in the blanks IRBNet #: Project Title: Association of Feeding Behavior and Gastric Function Principal Investigator s Name: Ivan Pavlov Date: A. Progress Report (to be completed if this application is being submitted as a triannual continuing review) A progress report must be submitted for the past approval period. The following information MUST be included: a) the exact number of animals used (do not approximate); b) # remaining and what is the disposition of the unused animals. What do you plan on doing with them? give them away (transfer) euthanize them or transfer them to the new application once it is approved. Don't forget to include this number on the total requested on the new application and justify their need on the new application along with any additional animals. c) Total new # needed, based on the number remaining and the protocol justification. d) a brief summary of results; and e) a discussion of unanticipated adverse effects. If none, so state. a. During the past year we tested 40 rats and 5 mice. b. I have 20 mice remaining in the DLAR facility. I do not have any rats remaining in DLAR. I intend to transfer the 20 mice to this new application once it is approve.

2 c. I need 120 mice and 40 rats to conduct my experiments this year remaining in DLAR = 100 mice, 40 rats. d. In studies with rats, we found that prior exposure to a food type elicits a conditional reflex response that includes salivation and excess gastric acid secretion. This study has been presented at the International Society of GI Physiologists, and has been submitted for peer-reviewed publication. In 5 preliminary studies with mice, we have evidence to support these same findings. e. There were no unanticipated effect in studies with the rats. In one mouse (the first trial) the externalized gastric tubing fell out. The mouse was immediately euthanized and DLAR was notified. A better method for securing the tubing was developed for the subsequent four mice, without unanticipated or adverse effects. B. Scientific Merit Review IACUC requires that the department chair (or chair of departmental review committee) conduct a scientific merit review. Is such a review conducted by another entity? Note that such review cannot be conducted by an agent with financial interest in the research, e.g. funding drug company. Yes (e.g., peer review funding agencies Such as (NIH,NSF, etc): No NIH NIGMS C. Personnel Experience With Animals 1) For each individual listed on the Registration Form as an investigator on this study, provide information below regarding experience for each of the listed procedures, for each species. If you have more than six personnel on your study, enter individuals on the IACUC Additional Personnel Form, which is available in the IACUC Designer Library within IRBNet. Download, complete, and then upload it into the package you are constructing at this time. 1. Name (First, Last):Pavlov, Ivan Phone: Monitor Handling Non-surgical Exp Methods Non-surv Survival Anesthesia (in Yrs. Blood Collect. Species 1: Rats Species 2: Mice Species 3: Species 4: 2. Name (First, Last):Fellow, Iam Phone: Species 3: Species 4: 3. Name (First, Last): Doe, John Phone Monitor Handling Non-surgical Exp Methods Non-surv Survival Anesthesia (in Yrs. Blood Collect. (in Yrs.) Species 1: Rats Species 2: Mice Monitor Handling Non-surgical Exp Methods Non-surv Survival Anesthesia (in Yrs. Blood Collect. (in Yrs.) Species 1: Rats Species 2: Mice Species 3: Species 4: Euthanasia Euthanasia (in Yrs.) Euthanasia (in Yrs.)

3 4. Name (First, Last): Phone 5. Name (First, Last): Phone 6. Name (First, Last): Phone Species 1: Species 2: Species 3: Species 4: Species 1: Species 2: Species 3: Species 4: T Species 1: Species 2: Species 3: Monitor Handling Monitor Handling Monitor Handling Species 4: *Attach separate sheet for additional personnel Non-surgical Exp Methods Non-surgical Exp Methods Non-surgical Exp Methods Non-surv Non-surv Non-surv Survival Survival Survival Anesthesia (in Yrs. Anesthesia (in Yrs. Anesthesia (in Yrs. Blood Collect. (in Yrs.) Blood Collect. (in Yrs.) Blood Collect. (in Yrs.) Euthanasia (in Yrs.) Euthanasia (in Yrs.) Euthanasia (in Yrs.) Occupational Medicine Requirement: All individuals listed on this study must be cleared by Occupational Health. All personnel listed must get an invitation from their P.I. to register in Occ. Health. The PI/personnel will enter the IACUC number to register with OH. If this is a brand new protocol, you will need to enter your IRBnet number to register your personnel. For questions regarding Occ. Health, contact Preventative Medicine at , leave a detailed message and someone will return your call. The link for Occupational Health information/updating/etc is: PI Occupational Health Assessment Exemption: If the PI wishes to be exempt from Occupational Health approval, the PI must check here: Checking the box means that the PI must refrain from all involvement with research animals in this study. However, the PI remains responsible for the proper care and use of the animals. Approval of this application cannot be granted until personnel have been cleared by the Department of Preventative Medicine. Allowing unauthorized personnel to enter DLAR or to participate on the study is a violation of policy. All personnel must have their own security code to enter DLAR and must code themselves into DLAR. Do not share your code or allow others to enter with your code, even if you are accompanying them. Sharing codes is a violation of DLAR policy. 2) Who will be responsible for training untrained individuals? Please contact the DLAR to arrange training and/or consultation, free of charge, for new personnel or for procedures novel to the applying laboratory. Include this individual in the personnel list (Section C).

4 Name: Ivan Pavlov (rats or mice) or Ima Fellow (rats only) Department: Medicine A min of 2 years experience will qualify these individuals to train Years experience in procedures on species listed: others. 3) Alternate contact who will assume animal care responsibility if the PI is unavailable: Name: Joe Colleague Dept:Medicine Campus Address: HSC T Telephone: Fax: Address: jcolleague@sunysb.edu D. Project Summary 1) Project Justification: In lay language (i.e., 8 th grade level), provide a brief overview of the aims/objectives of this study. Include a public description of the potential value of the study with respect to human or animal health, the advancement of knowledge, or the good of society. Avoid scientific jargon; scientific description belongs in Section H. The field size below will adjust to the user s input. People and animals have several levels of automatic responses to events or situations. In general the automatic responses are based on past experience, but the level of learning can be either conscious (where the animal is aware of the conditioning or training - i.e., given a treat for good behavior), or unconscious (where the response happens automatically as a reflex, and the animal has no conscious control over the response). In this study, we examine the unconscious automatic reflexes that occur in response to exposure to foods. These responses are "learned" by prior exposure to feeding. They are automatic, that is, the animal is not consciously making these responses happen. The responses are conditional, that is, without prior exposure to those specific foods, there is no reflex response. We are testing the responses to feeding as a model for conditional reflex responses in order to distinguish conscious and unconscious reflexes. One goal is to link conditional reflexes to gastric acid secretion and satiety hormone levels, to relate normal vs obese animal responses. Specifically, we are testing whether the conditional reflex responses to feeding differ in normal vs obese animals. 2) Check the appropriate statement: This research activity does not duplicate previous work. OR This research activity duplicates previous work. Justification must be provided below: E. Rationale and Alternatives to the Use of Animals 1) USDA regulations require that investigators explicitly consider alternatives to the use of animals for their research. Please provide a) a brief rationale for why animals are required for this study and b) why each of the proposed species is the most appropriate for this study: a) This study examines a complex integrative response that requires an intact animal system for study. The responses could not be obtained in cell culture, because they rely on the intact system.

5 b) Rats are a widely accepted animal model for conditional reflex responses, and thus we continue to use the rat model in order to relate our responses to the growing body of information in this area. The mouse genome is better known (vs. the rat) and several obese mouse models are available. 2) Provide a written narrative of your CURRENT literature search (within the last 3 months) that addresses alternatives to animal use and alternatives to any painful and distressful procedures. Specify the source, date of search, years covered by the search, and key words (key words MUST include the procedure being researched and alternatives). For example key words could include: thoracotomy, sternotomy, micropuncture, computer simulation, animal testing alternatives, animal welfare, etc. The IACUC recommends using standard database sources (e.g., MEDLINE) for crossreferencing key words from your most recent grant or publication with appropriate terms such as cell culture etc. Animal alternative resources are located at: 2A. Date of Search: Years Covered: Source:PubMed Keywords: conditional reflex response, gastric acid secretion, gastric cannulation leptin, obesity, rats, mice, alternatives Please provide a narrative of the search results: The search returned a total of 1125 articles on conditional reflex responses, 3098 on gastric acid secretion, on leptin and on obesity. Using logical combinations to search, conditional reflex responses + leptin returned 25 articles that showed the rat model is the primary accepted animal model, with 3 very recent articles demonstrating success in mice. Searching for conditional reflex responses + obesity returned 12 articles showing that these responses have been investigated in obese dogs, pigs and rats, but not yet in mice. Searching leptin + obesity returned 55 articles, of which 40 were clinical studies in obese patients with high peptide levels, and 15 articles in mice, relating peptide levels to degree of obesity in diabetic models. 2B. Based on the search conducted above: The PI certifies that alternatives to the use of animals and the procedures to be performed in this project are not available. The following alternatives to the use of animals and/or the proposed procedures have been identified but were deemed inadequate for the following reasons: 3) In the registration form, you are asked general information on the categories of research being conducted on each species. Please confirm below the category or categories which apply to this project and provide the additional information where requested. For Category C, detailed consideration of alternatives and justification is required. The number of animals in each category is tabulated in Section F below. Category A Category B Category C No Pain or Distress Relieved Pain or Distress Unrelieved Pain or Distress Potential pain or distress relieved by pharmacologic, behavioral or other means. Any procedure that would cause more than momentary or slight pain or distress.

6 e.g., observational studies, non-invasive manipulations These studies will cause no pain or distress to the subjects involved. e.g., tranquilization/sedation, general or local anesthesia, postprocedural analgesics, behavioral conditioning to restraint or minor pain/stress, medical treatment of disease states These studies will cause pain or distress that will be relieved by appropriate means. e.g., chronic untreated disease states, pain research These studies will cause pain or distress that is not relieved (strong justification required in Registration form ). F. Number of Animals Requested for Year 1 (by category) Purchased or Collected: Bred: Field Study/ Observational Study Species A B C A B C A B C Rats 40 Mice 120 USDA regulations require explicit accountability for determining the number of animals requested. To document this, provide a scientifically based rationale for the number of animals to be used (e.g., show the number of animals per experimental group a table is suggested and desired statistical inference level). Each experimental group should be accounted for in the experimental design of Section H. Note that the annual numbers requested in the table above defines the maximum number of animals you may acquire for this project each year. If there are requirements for an optimal number that exceeds the minimum necessary, check the optimal number option below and justify the extra animals. 1) Justify the number of animals that will be used during the 1st year for this research: One generic protocol will be tested using 40 animals per protocol. Four groups of animals will be tested: 40 normal weight rats; 40 normal weight C57/bl mice; 40 C57/bl mice fed a high fat diet; 40 ob/ob (genetically obese that do not make leptin) mice. The generic protocol that will be tested involves the following four experimental conditions (with the exact protocols outlined in section I) with 10 animals per experimental condition. Groups of 10 animals will provide sufficient statistical power (.90), based on a 2-tailed paired t-test, to detect a difference of 25% between the conditioned and non-conditioned treatment groups. Three Chronic Animal Experimental Conditions: Control, Sham surgery, Externalized gastric tube One Acute Experimental Condition In total there are 4 conditions x 10 observations (animals) per condition x 4 animal groups (rats, C57 normal mice, C57 high fat mice, ob/ob mice), for a total of 40 rats and 120 mice. 2) Will you require additional animals?

7 NO in deriving the number of animals requested, the PI has judiciously used the smallest number of animals necessary to obtain reliable and verifiable results appropriate to the experimental design. YES the optimal number of animals needed exceeds the minimal number of animals necessary for the reasons given below: 3) Will you be breeding rodents for this project? NO YES describe below (consult with DLAR for help with this): Rodent breeding requirements: Ahe IACUC s role for oversight regarding breeding colonies includes: ensuring that the need for a breeding colony has been established based on scientific or animal welfare concerns, that the procedures used in the breeding colony are evaluated and approved by the IACUC on a regular basis (e.g., as part of the semiannual program review), that there is a mechanism for tracking animals, and that the standards of care and animal well-being for the animals in the breeding colony are consistent with the Guide Investigators must develop a general health profile of a given strain that is relevant even to nonexperimental animals. You must consider potential adverse effects of each genetic model that you are requesting to breed. a) Briefly justify the need for establishing and maintaining a breeding colony in house as opposed to purchasing animals from an established vendor. b) Justify the number of animals needed to maintain the breeding colony for a year. c) Complete chart for each strain /line breeding. i. Number of breeders needed to establish a breeding colony: # female breeders *GALP - CRE(2) # times replaced Total # female breeders (Include original females # litters Produce for each female Total # of litters # pups Per litter Total # of pups produce d # male breeders # times replaced Total # of male breeders (include original breeders Total # of breeders Total # of animals

8 THE FIRST LINE ABOVE IS AN EXAMPLE ONLY Total number of breeders: 12(10 females+2 males) Total number of pups produce: 240 Total number of animals: 252 iii. ii. After completing the chart WITH YOUR NUMBERS, include your totals in the chart location in section G (number of animals requested for year) Be sure to include offspring that cannot be used in experimental procedures because they are the wrong sex, age, or genotype. The estimated numbers should clearly identify the following: Breeders Young that cannot be used because they are the wrong sex or genotype Animals that will be subjected to experimental manipulations d) Husbandry for breeding colonies: Who will be breeding/weaning the animals? DLAR DLAR colony manager Lab Which breeding system will be used? Time mating Monogamous pairs Tri breeders other: explain: Animal identification: Ear tag Tattoo Cage card Other e) When will the pups be weaned? By age 21 days Over age 21 days If weaned after 21 days of age, you will need to separate females before giving birth to avoid overcrowded cages. Used prior to or immediately after birth f) Genotyping and other procedures: Category. B animals Tail clipping (pups over 21 days of age require anesthetic Anesthetic used

9 Blood sampling Describe procedure used Other Anesthesia Age of genotyping: 0-14 days days 21 or older g) Are there any adverse health effects expected due to breeding genetic modified mouse models such as: seizures, paralysis, and increase output of urine? Yes No If yes, briefly give a general health profile and indicate special care needed: Caging Additional bedding changes due to increase urinary output Food/water Medical care h) Some genetically modified animals are created to develop a disease spontaneously. Others can develop debilitating health issues that were not the intended outcome. Please indicate humane endpoints in case of severe debilitating phenotypes. G. Animal Housing 1) If your registration form indicates live animals are taken out of DLAR for procedures, will it be for 12 hours or longer? No Yes If yes, download the Long-Term Animal Housing Form (available in the IACUC Designer Library in IRBNet), complete, and upload for inclusion with this submission package. 2) Will the animals be housed singly? No Yes Must provide strong scientific justification for this request below Animals must be kept in individual cages. Chronically instrumented animals must be kept in special cages with restricted movement. Specific animal chow is required for one group of normal C57/bl mice; this chow contains high fat. The group treated with Bigmacic Acid should be handled with care as the material can shed into the animal waste and must be disposed of as hazardous waste. 3) Is special housing or special animal care (i.e., isolation) required for the proposed activity or animal population?

10 No Yes describe below (prior consultation with the Director of DLAR required): See above. 4) Are the animals transgenics, knockouts or immuno-compromised? No Yes List Strain(s): Ob/ob mice Please note: Registration with the IBC is required for transgenic and knockout rodents which require BSL1 containment. The Transgenic/Knockout Animal Registration Form can be obtained in the IBC or IACUC Designer Library within IRBNet. If applicable, download this form, complete it, and upload/submit it with the study package you are constructing at this time. Approval by the IBC is required for any transgenic rodent models with BLS2 containment requirement and any non-rodent transgenic experiments. Full submission to the IBC is required. You can download and complete the Application for Approval of Recombinant DNA Activities from the IBC or IACUC Designer Library. You can upload the application into the study package you are constructing at this time for the IACUC, and when ready to submit, do so to both the IBC and the IACUC. Be sure the registration document associated with this study reflects both IACUC and IBC involvement. Directions are available in the IRBNet Instructions here: H. Experimental Procedures Using Animals 1) Standard Procedures: If the studies include any of the standard procedures identified below (click here for details of each), check them off here, give a general description of the experimental design in question H.2 below and account for the ultimate disposition of animals in Section O. Procedures should be mentioned in question H.2 below, but specific detail for the defined standard procedures is unnecessary, except for deviations which must be documented and justified in question 2) below. Check all that apply: Calorimetry Measurements in Rats and Mice In Vivo Micro CT (uct) of Rodents Monoclonal Antibody Production in Laboratory Animals In PI s Lab OR Use of Hybridoma / Cell Culture Facility Dept. of Microbiology and Molecular Genetics AND Alternative in vitro methods have been considered and were deemed inappropriate. [In Vitro Ascites Production] InVitro Ascites Production an alternative to the use of animals

11 Polyclonal Antibody Production Use of Freund s Adjuvant Production of Subcutaneous Tumor in Rodents Retroorbital Bleeding of Rodents Collection of Oocytes from Frogs Production of Transgenic Animals In PI s Lab Off-campus service; specify: On-campus service; specify: Gonadectomy in Rats and Mice Tail Biopsy Telemetry Unit Implantation in Rodents Telemetry Unit Implantation in Large Animals Field Research Observation of animals only (no physical contact and no manipulations of the environment that could have adverse effects on the animals). Limited physical contact or manipulation of environment (e.g., darting for collaring, netting, collection of samples). Describe the extra-observational procedures in Section H.2 below, and monitoring/management of potential adverse effects in Section I. Skip Section O. Ultrasound Imaging of Rats and Mice Implantation of Drug Delivery Systems in Rodents Vascular Perfusion Fixation (of mice and rats) 2) Experimental Procedures: Begin the narrative below with a brief (one or two paragraphs) scientific description of the overall experimental goals and project design. The description should provide a sequential overview of all experimental procedures and should account for each subject (by experimental group). The initial overview should be followed by a cookbook description of all experimental procedures related to the care and use of animal subjects. The description should assume that the reader is scientifically knowledgeable, but not an expert in your specific field of study. Do not paste in methods segments from grant applications that are not relevant to the care and use of animals, or information pertaining to in vitro work. Include the following: a) Duration of each procedure and the total time from initial contact to end of use for a single animal. b) Describe all drug manipulations, surgical procedures, scanning procedures, dietary or water intake manipulations and the route, amount, and frequency of blood collection, etc. Note: specific tabular information on drug doses and radiological or bio-hazardous substances are dealt with in separate sections below, as is specific information relating to survival surgery, anesthesia, and euthanasia. The experiments are designed to determine the relationship between obesity, leptin, gastric acid secretion (GAS) and conditional reflex responses. We will thus use several models of animals, and will obtain direct measures of rate of gastric acid secretion, leptin hormone levels, and rate of saliva production with exposure to feeding paradigms. Acute animal model. To determine basal secretion of saliva, leptin and GAS, these parameters will be determined in the anesthetized animals. Identical procedures will be performed on rats and mice; only the anesthetic dosages will differ. In anesthetized animals (see Anesthetic Section L), a tracheostomy is performed via a mid-line neck

12 incision. A right femoral arterial catheter is placed, using a cut-down technique; this is for blood collection to determine circulating leptin levels. A gastric tube is placed down the esophagus into the stomach for gastric acid collection. A suction tubing with slight negative suction is place in the oral cavity for saliva collection. Over a 2 hour period, gastric acid and saliva are collected each 15 minutes, and 0.1 ml blood is collected each 1 hour (total of 0.3 ml blood, which is <1% of the rat blood volume and 17% of the mouse blood volume total, or ~5% of the mouse blood volume). These animals are thus not conditioned, and not chronically instrumented. Chronic animal model. Following a 2 week period of free access to the test animal chow, the animals will be conditioned to feeding cycles by using a bell to preceed access to food. 120 animals will be conditioned to feeding over a 2 week period: 30 rats and 90 mice. Food will be withheld for 11 hours, and access to food will be preceeded by a bell. Food access will be permitted for 1 hour. Two feeding cycles will be permitted each day, each preceeded by the bell. Water will be given ad lib. Note that in prior studies, rats with this type of restricted food access do not show weight fluctuations (Nature Medicine, year, volume page). Likewise, in our preliminary studies in mice, weight did not fluctuate. After a 2 week conditioning phase, animals will be subdivided into one of four groups: control conditioned but not chronically instrumented, sham surgery, chronic nasogastric tube, externalized gastric tube. Ten rats and 10 of each mouse type will be assigned to each of the three groups. After two more weeks of continuing preconditioning, these animals will be euthanized. Control, No surgery. These animals are conditioned, but not instrumented. They will undergo the acute animal model procedures at the end of the additional two week preconditioning period. This group will thus test whether conditioning affects basal leptin, gastric acid and saliva secretions. Sham surgery. This control is essential to determine whether placement of the externalized gastric tube alone affects the outcome. In anesthetized animals, within a sterile surgical field, the abdomin will be shaved clean and disinfected with betadine and alcohol. A 0.5 cm incision will be made in the upper left quadrant of the abdomen, through the skin, muscle and fascia. The stomach will be punctured with a 27 g needle, and then the puncture site will be sutured using sterile 2-O silk suture. The fascia, muscle and skin layers will be progressively closed. A femoral artery catheter will be placed, using a cut down technique, as follows. the right inguinal area will be shaved clean and disinfected with betadine and alcohol. A 0.5 cm incision will be made from a point midway between the ilium and pubis, extending along the femoral sheath. The artery will be cannulated with an indwelling PE10 catheter, and secured to the surrounding muscle tissue with sterile 2-O silk. The catheter will be chased beneath the skin dorsally to a small hole at the nape of the neck. The tubing will be secured in place with a suture in the skin. Antibiotic ointment will be placed on the wounds. The animal will be monitored until recovery from anesthesia, and will receive pain relief (Section M). Externalized gastric tube. The Sham procedure will be followed, excepting that following stomach puncture, a thin polyethylene tubing (PE10, drawn to an external diameter of 0.05 cm) will be inserted into this hole. One suture with 2-O silk will secure the tubing at the site of entry, also closing the wound. A second suture will secure the

13 tubing to the wall of the stomach, approximately 0.5 cm away from the puncture site. As the fascia and muscle layers are closed, the tubing will be stitched around to further secure it. The tubing will be chased beneath the skin dorsally and will exit via a small hole at the nape of the neck. The femoral arterial catheter will be placed as for the Sham Control. The tubings will be labeled, and secured to the skin. Antibiotic ointment will be placed on the wounds. The animal will be monitored until recovery from surgery, and will receive pain relief. Chronic measures in the Sham and Externalized gastric tube groups. The conditioning with 2 feeding cycles will be continued in these, and the Control, No surgery group. For the sham and gastric tube groups, preceding each feeding cycle (1x / day), the animal within the special cage will be weighed, and 0.1 ml blood and gastric acid will be collected by 5 minutes after the bell, yet before feeding is permitted. Collection should take not more than 60 seconds. Following gastric acid collection, the tubing will be flushed with saline. Following blood collection we will inject 0.2 ml saline to replace the volume taken, and the arterial catheter will be flushed with heparinized saline to keep the catheter clear. This will be <1% of the rat blood volume, and ~5% of the mouse blood volume. At the end of two weeks, the animals will be euthanized. I. Adverse Effects: Monitoring and Management (IMPORTANT NOTE: ANY unexpected adverse events or unexpected loss of animals must be reported to DLAR Vet at time of occurrence). 1) Explicitly describe the potential adverse effects of each experimental procedure conducted on, and all agents administered to, the animal subjects. For each item, include a statement on how the problem will be clinically managed, if it occurs. Acute experimentation. The chief potential adverse effect is the animal awakening unexpectedly from anesthesia. Although anesthesia level is checked and documented q 15 m, there is a potential of early emergence. Animals that awaken (deliberate movements to "right" themselves) will be given supplemental anesthesia. Chronic model. Following recovery from this procedure, the animals could become sick from sepsis (due to stomach contents leaking into the peritoneum), the gastric tube could slip out, the arterial catheter could slip out, or the wounds could become infected. If they become sick and sepsis is suspected, clinically they will show ruffled fur, poor nest making ability, lethargy and weight loss. If sepsis is suspected, the animals will be euthanized with anesthetic overdose, and the abdominal cavity will be opened and inspected; DLAR will be notified of our findings. If the gastric tube slips out, the animals will be euthanized immediately. If the femoral arterial catheter slips out, the animal will likely hemorrhage within minutes; this would be reported to DLAR. If the catheter is found out, and the animal is alive, it will be euthanized immediately. If the wounds become infected (abdominal wound, nape of the neck wound) they will be first cleaned with betadine, and treated with antibiotic ointment for 24 hours, and DLAR will be notified. If the infection does not improve, and the DLAR staff has no further

14 recommendations for treatment, then the animals will be euthanized. 2) For the procedures above that might cause pain, discomfort or distress, indicate which of the following clinical parameters will be monitored (check all that apply): Activity Appearance Respiratory rate Movement Urine/feces Grooming Body temp Vocalization Posture Weight loss Behavior Appetite Heart rate Other (specify): Lab Tests (specify): 3) Specify the frequency of monitoring (e.g., twice per week, once per hour): Chronically instrumented animals, twice daily: appearance, activity, posture, grooming, weight, behavior. 4) What conditions and/or complications would lead to removal of an animal from the study (an early endpoint )? In I.1 specific endpoints are noted. Additionally, any animal losing 15% of their body weight will be immediately euthanized. J. Survival, Manipulations or Procedures Will any animals recover from anesthesia after surgery or manipulations/procedures? (Detailed description of all surgical procedures/manipulations should be provided in Section H.) n/a No Yes complete questions 1-3 below: 1) Is the surgery, manipulation or procedure: Minor Major (penetrates a body cavity or can cause permanent physical or physiological impairment such as fracture, etc) 2) For surgical procedures, manipulations or procedures, if the primary/senior surgeon for the study has less than 2 years surgical experience in the species proposed, check here and contact the DLAR for consultation and to arrange observation of the initial surgery. 3) How long will animals survive after the surgery or manipulation? 2 weeks 4) List the post-surgical analgesics to be used. If analgesia will be withheld, strong scientific justification is required below. Buphenorphin will be used on both the rats and the mice post-op. 5) Will multiple survival surgeries, manipulations or procedures be performed on the same animal? No Yes complete the following questions: Species: Number of animals: Time interval between surgeries or

15 procedures: Provide scientific justification for multiple surgeries or procedures on the same animal: K. Anesthesia Will any of the proposed procedures involve anesthesia? No Yes; complete questions 1-5: 1) Pre-anesthetic fasting (hours): 2) Pre-anesthetic medications: Species Agent Dose* Max Vol. Route * For injectable agents, provide dosage in mg/kg or other appropriate units. 3) Anesthetics: Species Agent Dose* Max Vol. Route Freq. of Admin Rat Pentobarbital ml i.p. once sodium mg/kg Mice Pentobarbital sodium 75 mg/kg 0.2 ml i.p. once * For injectable agents, provide dosage in mg/kg; for inhalants, give % in O 2, or other appropriate units 4) Monitoring of anesthesia (check all that apply): Corneal reflex Heart rate Respiratory rate Blood pressure Response to noxious stimulus Other (specify): Frequency of monitoring: Every 15 minutes 5) Monitoring of recovery from anesthesia (survival procedures) or procedures: Heart rate Respiratory rate Body temp Response to noxious stimulus Other (specify): Frequency of monitoring: Q 15 min Estimated time of recovery until animal is ambulatory: 1 hr. L. Administration of Substances Other than Anesthetics 1) List all pharmaceutical, therapeutic and experimental agents (e.g., analgesics, antibiotics, paralytics, infectious agents including bacteria or viruses, experimental drugs). If a paralytic is used, clearly justify its use in Section H (Experimental Procedures Using Animals). Species Agent Dose * Max Route Frequency Duration of Drug Action Vol. Rat Buphenorphi ne ml SC Once, then PRN 8-12 hours

16 Mouse Rat Rat/mouse Buphenorphi ne Bigmacic acid Triple antibiotic oint ml SC Once, then 8 12 hours PRN 1 Oral Daily 24 hours 1 Topical Daily as needed 24 hours * provide dosage in mg/kg or other appropriate units M. Prolonged Restraint of Conscious Animals 1) Will any of the proposed procedures involve restraint of a conscious animal for periods longer than required for brief physical examination, routine blood collection, or injection (e.g., study carts, chairing apparatus, Bollman cages)? No Skip to Section N. Yes answer the questions below and continue with Section N. 2) Provide scientific justification for the need to restrain unanesthetized animals: Acute experimentation. The chief potential adverse effect is the animal awakening unexpectedly from anesthesia. Although anesthesia level is checked and documented q 15 m, there is a potential of early emergence. Animals that awaken (deliberate movements to "right" themselves) will be given supplemental anesthesia. Chronic model. Following recovery from this procedure, the animals could become sick from sepsis (due to stomach contents leaking into the peritoneum), the gastric tube could slip out, the arterial catheter could slip out, or the wounds could become infected. If they become sick and sepsis is suspected, clinically they will show ruffled fur, poor nest making ability, lethargy and weight loss. If sepsis is suspected, the animals will be euthanized with anesthetic overdose, and the abdominal cavity will be opened and inspected; DLAR will be notified of our findings. If the gastric tube slips out, the animals will be euthanized immediately. If the femoral arterial catheter slips out, the animal will likely hemorrhage within minutes; this would be reported to DLAR. If the catheter is found out, and the animal is alive, it will be euthanized immediately. If the wounds become infected (abdominal wound, nape of the neck wound) they will be first cleaned with betadine, and treated with antibiotic ointment for 24 hours, and DLAR will be notified. If the infection does not improve, and the DLAR staff has no further recommendations for treatment, then the animals will be euthanized.

17 3) Describe the type of restraint to be used: The cages to be used are clear plexiglass that restrict animal turning, and thus are the width of the animal. They are sufficiently long so that the animal can move forward and backward. 4) Specify the duration and frequency of restraint: Two weeks, continuously 5) How frequently will animals be monitored during restraint and what will be monitored? N. Food/Water Restriction 1) Will any of the proposed procedures involve restriction of free access to food or water? No Skip to Section O. Yes answer the questions below and continue with Section O. 2) Specify the restriction protocol (e.g., food restriction to 85% normal body weight, water restriction as a precursor to rewards for behavioral performance): Water will not be restricted. Food will be provided for 1 hour during each 12 hour period. 3) Provide scientific justification for the restriction: Food restriction is part of our experimental paradigm. We are conditioning the animals to anticipate food when they hear a bell, and are measuring the responses to that conditioning reflex. 4) Specify the time period of the restriction: Food will be restricted for 4 full weeks for 30 rats and 90 mice. 5) Specify the steps to ensure adequate nutrition/hydration and the variable to be monitored to ensure animal health during the restriction: Animal weight and behavior will be monitored 2 x daily. O. Disposition of Animals Check whether euthanasia or an alternative to euthanasia will be used in this study and provide the additional information as directed. Note: Investigators are responsible for the euthanasia of animals in their research.

18 Euthanasia as an endpoint 1) Describe the method of euthanasia for each species or procedure. NOTE: Decapitation, cervical dislocation, etc. without anesthesia requires strong scientific justification. Pentobarbital sodium, i.p., 100 mg/kg for rats or mice. 2) Euthanasia agents List pharmaceutical agent(s) and their dose(s) used during/for terminal anesthesia/euthanasia. Species Agent Dose Route Rats Pentobarbital sodium 100 mg/kg i.p. Mice Pentobarbital sodium 100 mg/kg i.p. Alternatives to euthanasia (adoption, transfer, etc.) 1) If animals will not be euthanized at the end of the project, describe what will happen to them. If the animal(s) could be of potential use to other investigators or be used for training purposes, please contact the DLAR Office. P. Hazardous Substance Use in Animal Research 1. Are genetically engineered materials (e.g., transgenic animals, etc.), and/or is recombinant DNA technology, used in the course of this study? no Proceed to P.2 yes Are only transgenic animals involved? no Institutional Biosafety Committee (IBC) #: If you do not have a current IBC#, see below for full submission to IBC information yes Transgenic rodents requiring BSL1 containment? yes Registration with the IBC is required: The Transgenic/Knockout Animal Registration Form can be obtained in the IBC or IACUC Designer Library within IRBNet. If applicable, download this form, complete it, and upload/submit it with the study package you are constructing at this time. All acquired or created genetically modified rodents must be registered or approved by the IBC (this requirement extends to the creation of new strains through breeding of existing transgenic strains with other transgenic -or knockout- and wild type rodents). no For transgenic non-rodent experiments, or transgenic rodent models requiring BSL2 containment, Approval by the IBC is required. Full submission to the IBC is required. SEE BELOW You can download and complete the Application for Approval of Recombinant

19 DNA Activities from the IBC or IACUC Designer Library. You can upload the application into the study package you are constructing at this time for the IACUC, and when ready to submit, do so to both the IBC and the IACUC. Be sure the registration form associated with this study reflects both IACUC and IBC involvement. Directions are available in the IRBNet Instructions here: 2. Will a potential hazard to humans or other animals be introduced in the course of this live animal research/educational activity? No Yes consult with the Department of Environmental Health and Safety at SBU. Then complete this section and provide a copy of the Radiation or Biohazard Safety Precautions to be posted during the study activities. Specify methods for safe and proper disposal of all contaminated materials. a) Type of Hazard: Dose and Route Radioisotope Hazardous Chemicals/Carcinogens, Bigmacic acid (1 per day orally) toxic, corrosive, reproductive haz, etc. Live Microorganism Parasite Gamma Cell Irradiator Use X-Ray, PET, CT Scan Radiopharmaceuticals (scans) Other 1. For each hazard selected, attach an MSDS form or other appropriate documentation of the hazard and proper handling precautions. Also include information on all vectors and agents in the box below and provide strain designations. Bigmacic Acid is a known hazard. It causes obesity in lab animals and is a suspect agent for the cause of diabetes, red hair and other diseases. The laboratory staff is well versed in the hazards associated with this material and is equipped to handle the unique hazards associated with this material. 2. If a license or permit is required to use the above hazard, provide details (permit/license #, agency) and submit copy: b) Affected Animal Species Max. Daily Census of affected animals Rats Is special housing or animal care required due to hazardous substance use? No Yes Include this information in Section G. Animal Housing a. Where will animals be housed during exposure? Animal Facility(DLAR) Lab/room Other

20 If lab/room or other, how long will they be house there? b. Where will animals be housed after exposure? Animal Facility(DLAR) Lab/room Other If lab/room or other, how long will they be house there? 4. Live microorganism or chemical agent information (check all that apply): Biocontainment Required: BSL1 BSL2 BSL3 (for biological organisms only) a. Housing Requirements: Biosafety Cabinet Required: Date of Last Certification of cabinates located outside of DLAR: Ventilator rack/microisolator cages Negative air pressure room (Biohazard Space) Other b. Hazard to: Personnel Other Animals 5. Amount of hazardous/radioactive material released after dosing (consult with Environmental Health and Safety if unknown): Urine Skin Feces Aerosol/expired air other Time frame for secretion/excretion/infectivity of above: 6. Means of personal exposure from radioisotopes, carcinogens or other toxic/noxious substances: Not Transmittable Ingestion Aerosol Accidental Infection Skin/Mucus Membrane Contact 7. Personnel safety precautions required: Surgical Mask Respirator- type: required for respirator use Disposable Gowns Shoe Covers Disposable Caps Disposable Coveralls Disposable Gloves Other: (i.e. N95, chemical cartridge) Note clearance Use of an effective disinfectant for the agent or autoclaving is required to render all cages and other equipment used with infectious agents at BSL2 (or greater) containment safe for reuse or disposal. All waste and bedding from BSL2 (or greater) containment must be disposed of as Regulated Medical Waste (red bag) 8. Methods for disposal or decontamination of carcasses, equipment and waste that are contaminated: Animal Equipment (bottles, wire bars, shoe box cages, iso lids, etc) Normal processing Autoclave before normal processing

21 Bedding/Waste Carcass Chemical decontamination/disinfection Hold for EHS Normal disposal Red bag and send out as RMW(incinerate) Hold for EHS Hold for Radiation Services Red bag and send out as RMW(incinerate) Hold for EHS Hold for Radiation Services Please note: MSDS forms pertaining to this study must be posted where the work is being done, along with the approved application/amendments so personnel have quick access to them. Certifications Principal Investigator: Your e-signature associated with this submission means that you agree: 1. To execute the research project/educational activity as described in the application once approved, and in subsequent revisions required as a result of IACUC review. 2. To request approval from the IACUC for changes in procedures described in the application, should they become necessary. 3. To maintain continued approval with IACUC for active studies. 4. To comply with the federal, state and campus regulations with regard to the humane treatment of animals, radioisotope/biohazard safety, and other issues covered by applicable federal, state and campus regulations. 5. To assure that all individuals conducting the procedures described are appropriately trained or to make provisions for training prior to using animals. 6. To be responsible for the supervision of staff conducting the procedures described in the application. 7. To consult with DLAR clinical veterinary staff to ensure appropriate veterinary care. (Note that if you, or your responsible support staff cannot be contacted, and animals show evidence of illness or pain, emergency care, including euthanasia, may be administered at the discretion of the veterinary staff.) 8. To ensure that all personnel listed on this application will read and understand the contents of this protocol before beginning any procedures on research animals. The approved application and any amendments to it must be posted where the animal work is being conducted so personnel can easily refer to it. Your e-signature also assures that your co-investigators are fully cognizant of the details of this submission, will conduct all aspects of the study as approved by the IACUC and have been informed of potential hazards, safe work practices, availability of medical surveillance, and training related to this project. Department Chair*:

22 Your e-signature associated with this submission means that you have read the documents contained within, and the project, as described has your endorsement as scientifically meritorious. *Note: If the department chair or member of the departmental review committee is an investigator on this study, s/he can electronically sign as PI if his/her role is as principal investigator, but s/he cannot additionally sign this certification as the chair or member of review committee. The preferable signatory is the Chair (if the investigator is on the review committee), your Dean (if the investigator is the chair), or the VP for Research (if the investigator is Dean). Official designees of these signatories are also acceptable so long as the designee is not a subordinate to the investigator in any way.