表1 動物別(中英文)及品系(不同項目請分欄列出) 表2 動物來源(不同項目請分欄列出) 一 、 小 鼠 Mice/BALB/c 小鼠 十、迷 你 豬 廿六、蛇 Mice/C57BL/6 小鼠 十一、豬 廿...
21.6 (Rev. 5/02 js) PROTOCOL # REQUEST FOR AUTHORIZATION TO PERFORM RESEARCH UTILIZING LIVE ANIMALS Please provide the requested information for each type of experiment. Use additional pages for Section 2. Answer all questions fully and completely. Insufficient information may result in delay of the review process. The AWC reserves the right to request additional information. Submit original to Joanne Stocker (Virology Dept.) at least two weeks before the start of experimentation. Keep one copy of the protocol for your records. ________________________________________________________________________________________ __ This application is (circle one): (a) New (b) Review or update of Protocol # ________________________________________________________________________________________ __ SECTION 1 I. APPLICATION DATA Protocol Title: Effect of ME7 or 139A scrapie agent on NF?B knockout mice (B6.129P-Nf?b1tm1Bal) and B6129PF2/J mice Starting Date: 2/5/03 Projected Completion Date: Indefinite Xuemin Ye Principal Investigator: Secondary Investigator(s): Technicians/Students: Department: II. Virology FUNDING DATA Provide the information requested. A. B. Anticipated Funding Source: Status of Grant Application: Application submitted on Application to be submitted on NIH C. Signature Certification of Principal Investigator Date Title Signature certifies that the principal investigator will conduct the project in full accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, USDA rules and IBR regulations governing the use of live vertebrate animals for research or teaching purposes. It is understood that AWC approval is valid for a period of 36 months following the date of original approval and release with annual update required. It is further understood that should this project be submitted for external funding, the information presented on the Request for Authorization form reflects accurately the animal use in the full grant application. D. Signature Certification of Review by Student's Advisor (required for all protocols submitted by students) Date Title E. Certification of Chairperson of Animal Welfare Committee III. BIOHAZARDOUS MATERIAL: If the animal use involves biohazardous materials, the appropriate category should be checked and approval obtained from the appropriate committee chairperson. Infectious Agents X Toxic Compounds Radioisotopes ____________________________________________ Signed: Dr. Richard – Infectious Agents ____________________________________________ Signed: Dr. Richard J. – Toxic Compounds _____________________________________ Signed: Dr. Fred – Radioisotopes IV. CATEGORY OF RESEARCH: experimentation. The investigator should check the appropriate category(ies) of A The research involves either no pain or potentially involves momentary, slight pain, discomfort or distress. ● Includes simple invasive procedures (e.g., injection, blood sampling), terminal anesthetic surgery, collection of tissues preceded by standard euthanasia, behavioral testing without stress. B The research potentially involves minor short-term pain, discomfort or distress which will be treated with appropriate anesthetics/analgesics. ● Includes minor survival surgery with anesthesia and without significant post-op pain (e.g., biopsy), implantation of minor chronic catheters (e.g., femoral arterial and venous catheters, flow probes, etc.), short-term physical restraint (<60 min) of awake animals, induction of minor behavioral stress. C The research involves chronic maintenance of animals with a disease/functional deficit and/or procedures potentially inducing moderate pain, discomfort or distress which will be treated with appropriate anesthetics/analgesics. ● Includes major survival surgery with anesthesia and/or inducement of a functional deficit (e.g., orthopedic surgery on femur, amputation, bowel resection, cardiac surgery, adrenalectomy, non-painful tumor inducement, use of immunological adjuvants), physical restraint (>60 min) of awake animals, induction of more than minor behavioral stress. X D The research potentially involves pain, discomfort or distress (greater than that attending routine injection) which cannot/will not be alleviated through the administration of appropriate anesthetic, analgesic or tranquilizer drugs. ● Examples include pain research, radiation testing, toxicity testing and lifetime carcinogenesis experiments. ● In order for a protocol to qualify as Category A, B, or C appropriate anesthetics/analgesics must be used if the animal will experience more than momentary slight pain. Momentary slight pain is defined as pain no greater than the level and duration of pain attending a routine injection. Alternately, the animal must be immediately euthanized upon evidence of such pain or the protocol classified as Category D. V. in ANIMAL CHARACTERISTICS: the research project. The investigator should state the required number of animals to be used Species/Strain B6;129Ptm1Bal Nf?b1 B6129PF2/J Sex F or M F or M Age/Weight 1-16 mos 1-16 mos Animal Vendor Bred in house (Origin: Jackson Lab) Bred in house (Origin: Jackson Lab) Location of Housing Main Colony Main Colony Total Number 60 60 INSTRUCTIONS: The AWC requests the information described in the following section pursuant to its charge by the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals. Submit all information using the headings listed below in boldface type. Address each item independently, without reliance on information covered under other items. Subheadings (e.g., a, b, c, d) of points VII, IX, XII, XIII and XIV must be included and addressed in sequence. Include sufficient information to allow reviewers to judge whether the research merits the use of animals and whether the animals will be treated humanely. Do not submit major sections of your grant proposal or excessive detail of assays not related to the use of animals (e.g., chemical assays, molecular biology, in vitro tests). All abbreviations and terms not part of common usage should be clearly defined. Remember that not all the reviewers are familiar with your area of research. Consult the AWC Guidelines during completion of this form. Section 2 has a limit of 5 pages excluding references. SECTION 2 I. II. Purpose of the Study. State the specific scientific objectives (aims) of the research. Potential Value of the Study. State the potential value of the study with respect to human or animal health. Identify the information gaps the project is intended to fill. If the research duplicates previous experiments, explain why the duplication is necessary. If it does not duplicate previous research, this should be stated. Please provide documentation supporting your position. III. Alternatives to Animal Use. What alternatives to the use of live animals did you consider? What reasons did you have for rejecting them? If specific alternatives to live animals do not exist, this should be stated and justified appropriately. In general, a simple statement that there are no alternatives will not suffice. IV. V. Species Justification. State how you selected which species to study. Justification of the Number of Animals Requested Based Upon the Experimental Design. Provide detailed justification for the number of experimental and control animals requested. Include a brief description of the experimental design and state the number and species/strain of animals per group/subgroup in each experiment/procedure. A table or block design can facilitate the review of this section. VI. Previous AWC Approved Protocols. Does this project contain procedures which have been approved in previous protocol applications? If yes, please indicate the AWC approval number(s) and points of overlap/similarity with the present protocol. Do not omit material from this protocol because it was discussed in a previous protocol. Reviewers will not, in general, consult previous protocols. VII. a) Procedures. Describe the animal-related procedures by addressing the following in sequence under the subheading (a, b, c) indicated. This section must clearly reflect how the purpose of the study will be approached. Using boldfaced subheading identifying the procedures, describe sequentially with a reasonable degree of detail all procedures (surgical and non-surgical) to be carried out on live animals. When chemical agents are administered, specify the dose and route of administration. The end-points of procedures and the time frame must be clearly defined. For chronic as well as acute experiments, the length of time the animals will be maintained prior to euthanasia must be estimated. If live animals will be maintained outside of their normal housing facility for greater than 12 hours, identify why, where and how long the animals will be used at that location. If survival surgery will be performed, identify where the surgery and postoperative recovery will take place. b) c) VIII. Alternatives to Painful Procedures. If the research involves procedures that may cause more than momentary or slight pain or distress to the animal, the investigator must consider alternative procedures that cause less potential pain or distress. Address why alternative procedures either do not exist or were rejected. State the method(s) and/or sources that were used to determine that alternatives are not available and/or are scientifically unacceptable;
or If the procedure is acute, this should be so stated. IX. Restraints. Describe any proposed restraint (other than short-term hand-held restraint) to be used on awake animals by addressing the following in sequence under the boldfaced subheadings indicated. a) b) c) d) Justification. Justify the need for restraint. Description. Identify or describe the restraint(s). Duration. State the duration of the restraint period. Conditioning. Describe steps to be taken to condition the animals to the restraining device and assure the comfort and well-being of the animals;
or If restraints will not be used, this should be so stated. X. Pain Control During Procedure(s). For each procedure that causes more than momentary slight pain, identify the procedure, specify the preoperative preparation (e.g., antibiotics, tranquilizers, etc.) and specify the anesthetic(s) to be used during that procedure. Include dose (e.g., mg/kg) and route (e.g., IM, IV) of administration of all agents;
or If anesthetics are indicated but will not be administered, strong justification for withholding anesthetics must be provided in this section;
or If pain control during procedures is unnecessary, this should be stated and justified. XI. Estimation of Potential Postoperative/Intervention Pain. If a procedure (e.g., thoracotomy, administration of drugs, chemicals) or induced condition (e.g., tumor, pancreatitis) will potentially cause more than momentary slight pain, estimate the magnitude and duration of any pain, discomfort or distress the animals may potentially experience;
or If animals will not experience postoperative/intervention pain, discomfort or distress in association with any specific procedure(s) or condition, this should be stated and explained. XII. Post-Procedure/Chronic Care. Describe the proposed care of the animals by addressing the following in sequence under the boldfaced subheadings indicated. Alternatively, if post-procedure care is unnecessary, this should be so stated and justified. a) Post-Procedure Monitoring. State the frequency and length of time over which post-procedure examinations/monitoring of the animals will be performed. Criteria for Pain. State the specific criteria that will be used to measure/monitor acute and chronic pain. Analgesic(s). If it is anticipated that an animal may be subjected to more than slight pain, discomfort or distress, specify the analgesic(s) which will be used to prophylactically treat the animal. Include dose (e.g., mg/kg), route (e.g., IM, IV) frequency and duration of administration;
or If analgesics will not be administered routinely/prophylactically, state the specific criteria for administration of analgesics. In addition, specify the analgesic(s) including dose (e.g., mg/kg), route (e.g., IM, IV) and frequency of administration;
or If analgesics are indicated but will not be administered (Category D), strong justification for withholding analgesics must be provided in this section. Antibiotics. Specify any antibiotic to be used. Include dose (e.g., mg/kg), route (e.g., IM, IV), frequency and duration of administration. If antibiotics are not necessary, this should be so stated. b) c) d) XIII. Euthanasia/Disposition of Animals. Describe euthanasia of the animals by addressing the following in sequence under the boldfaced subheadings indicated: a) Method of Euthanasia. Specify the method(s) of euthanasia including the dose (e.g., mg/kg) of any injectable agents. Describe the criteria for determining that euthanized animals are dead. Simply stating that they will be put into 100% CO2 or the like is not sufficient. b) Criteria. Describe the criteria for euthanasia of the animals (e.g., end-point of experiment, specific time period, tumor size, etc.). Criteria for Premature Euthanasia. For Category C and D experiments, specify the criteria for premature euthanasia of the animals (e.g., significant pain or sickness, inability to feed, etc.);
or If animals will not be euthanized, state the final disposition. c) XIV. Investigator(s) Qualifications/Experience. For each individual listed in Section 1, describe the expertise and experience related to this project by addressing the following in sequence under the boldfaced subheadings indicated. a) b) Knowledge of Species. Address familiarity with characteristics of the selected species. Relevant Experience. Describe experience with regard to the specific procedures to be applied to live animals, methods of pain control, postoperative care and euthanasia. Responsibilities. State the individual's specific role/responsibility in this project. c) XV. XVI. References (optional). Provide a list of key references (maximum of five) that support the statements contained in Section 2 (II. Potential Value of the Study) of this form. Investigator Comments. Include any additional comments relevant to the proposed study and/or attach any relevant material the AWC should consider during the review process. IBR 21.6 (Rev. 8/92) PROTOCOL # _______ REQUEST FOR AUTHORIZATION TO PERFORM RESEARCH UTILIZING LIVE ANIMALS - CHECKLIST SECTION 1 _____ _____ _____ _____ _____ _____ _____ _____ _____ I. II. APPLICATION DATA FUNDING DATA A. Anticipated Funding Source B. Status of Grant Application C. Certification of Principal Investigator D. Certification of Review by Student's Advisor (required for all protocols submitted by students) BIOHAZARDOUS MATERIAL CATEGORY OF RESEARCH ANIMAL CHARACTERISTICS III. IV. V. SECTION 2 _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ I. II. III. IV. V. VI. VII. Purpose of the Study Potential Value of the Study Alternatives to Animal Use Species Justification Justification of the Number of Animals Requested Based Upon the Experimental Design Previous AWC Approved Protocols Procedures a) b) c) Alternatives to Painful Procedures Restraints a) Justification b) Description c) Duration d) Conditioning Pain Control During the Procedure(s) Estimation of Potential Postoperative/Intervention Pain Post-Procedure/Chronic Care a) Post-Procedure Monitoring b) Criteria for Pain c) Analgesic(s) d) Antibiotics Euthanasia/Disposition of Animals a) Method of Euthanasia b) Criteria c) Criteria for Premature Euthanasia Investigator(s) Qualifications/Experience a) Knowledge of Species b) Relevant Experience c) Responsibilities References (optional) Investigator Comments ______________________ Approval Date VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. _______________________________________________ David Ph.D., Chairperson, AWC SECTION 2 I. Purpose of the Study: The aims of this project are (1) to compare susceptibilities of mouse-adapted scrapie agents in NF?B knockout mice and its control B6129PF2/J mice;
(2) to assess the role of NF?B in the infectivity and pathological changes in scrapie-infected mice;
(3) investigate the potential treatment of NF?B pathway in prion diseases. II. Potential Value of the Study This study will increase our understanding of the mechanisms that underlie disease processes in transmissible spongiform encepholopathies (TSE) and thereby provide possible strategies for prevention and/or treatments of these diseases. By using scrapie-infected NF?B knockout mice, we will investigate the role of NF?B in scrapie pathology and incubation period. We will be able to determine if NF?B knockout mice have prolonged scrapie incubation period and decreased prion protein accumulation. We can also study the potential value of drugs in NF?B pathway in treatment of prion diseases. III. Alternative to Animal Use We will determine if NF?B knockout mice have prolonged scrapie incubation period and decreased prion protein accumulation. This can only be done with NF?B knockout mice. IV. Species Justification We will investigate the role of NF?B in scrapie pathology and incubation period, therefore we need NF?B knockout mice (B6.129P-Nf?b1tm1Bal) and its background control strain B6129PF2/J mice (Sha et al., 1995). V. Justification of the Number of Animals Requested Based Upon the Experimental Design We will use 10 mice for each group to obtain significant data on these parameters. Inoculate Method B6.129P-Nf?b1tm1Bal B6129PF2/J 139A i.p. 10 10 i.c. 10 10 i.p. 10 10 ME7 i.c. 10 10 i.p. 10 10 NMB i.c. 10 10 VI. Previous AWC Approved Protocols Not submitted before. None on this particular protocol, although there are protocols dealing with titration and pathogenesis of scrapie. VII. Procedures a) Animals will be injected with scrapie agent (139A or ME7) or normal mouse brain homogenize (NMB) as control by i.p or i.c. route (25 ?l), and then observed for clinical changes, which involves measuring the capacity of the animals to traverse a grid. Scrapie-positive animals have a staggering gait on the apparatus, tend to miss placing their feet on the rungs and when they do miss, they fail to replace their feet quickly. At the end of the incubation period, mice will be perfused following deep anesthesia induced by injection of 5 mg Nembutal i.p. Perfusion is used to ensure good quality sections for light microscopy and immunocytochemistry stain (Vaughan et al., 1981;
Ye et al., 1998). b) Not applicable (N/A). c) N/A. VIII. Alternatives to Painful Procedures There are no alternatives to the only known painful procedure, i.e., intracranial inoculation. Intracranial inoculation is the best route for yielding a reproducible sequence of parameters of agent-host interaction. IX. Restraints N/A a) Justification: N/A b) Description: N/A c) Duration: N/A d) Conditioning: N/A X. Pain Control During the Procedure(s) Ether during i.c. injection. XI. Estimation of Potential Postoperative/Intervention Pain Scrapie is a neurodegenerative disease characterized by a long asymptomatic incubation period followed by a relatively short clinical course that ends in death. Clinical signs include ataxia, which in some cases can proceed to the point where the animals are apparently incapable of voluntary movement. It may be difficult to discern pain in scrapieinfected animals. XII. Post-Procedure/Chronic Care a) Post-Procedure Monitoring: N/A b) Criteria for Pain: N/A c) Analgesic(s): N/A d) Antibiotics: N/A XIII. Euthanasia/Disposition of Animals a) Method of Euthanasia/Disposition of animals: if animals are sick prior to the end of the scrapie incubation period, they will be given an overdose of CO2 in a pre-gassed CO2 chamber. Death will be determined by failure to respond to a leg pinch and the absence of breathing. b) Criteria: mice will be euthanized at the end of the scrapie incubation period. c) Criteria for Premature Euthanasia: severe illness or injury indicating little chance of recovery. XIV. Investigator(s) Qualifications/Experience a) Knowledge of Species: Xuemin Ye, Harry C. Meeker and Richard I. Carp are extremely familiar with mice in general and with scrapie-infected mice in particular. b) Relevant Experience: Xxx Ye has approximately 14 years of experience with the procedures in the protocol. Richard I. has over 30 years of experience with mouse experimentation. Harry C. has more than 12 years of experience with mouse experimentation. c) Responsibilities: Xuemin Ye will have overall responsibility for the experiment and will do some of the perfusions and organ harvesting and immunostaining. Harry C. will score the mice, do some of the perfusion studies and will help with organ harvest. Dr. Carp will observe the health status of the mice and assist in scoring for scrapie. XV. References (optional) 1. Vaughan JE, Barber RP, Ribak CE, House CR (1981): Methods for the immunocytochemical localization of protein and peptide involved in neurotransmission. In: Johnson JE (ed.) Current Trends in Morphological Techniques, Vol. III. CRC Press. Florida, pp. 33-70. 2. Sha WC., Liou HC., Tuomanen EI., and Baltimore D. (1995). Targeted disruption of the p50 Subunit of NF-?B leads to multifocal defects in immune responses, Cell, 80:321-330. 3. Ye X., Scallet AC., Kascsak RJ. and Carp RI. (1998). Astrocytosis and amyloid deposition in scrapie-infected hamsters, Br. Res. 809:277-287. 4. Kim JI., Ju WK., Choi JH., Kim J., Choi EK., Carp RI., Wisniewski HM. And Kim YS. (1999). Expression of cytokine genes and increased nuclear factor-kappa B activity in the brains of scrapie-infected mice. Mol. Br. Res. 73:17-27. XVI. Investigator Comments NF?B is a heteradimeric transcription factor composed of p50 and p65 subunits. It can be activated in many cell types and is throught to regulate a wide variety of genes involved in immune function and development. It has been found that increased NF?B acitivity in the brains of scrapie-infected mice, which may contribute to the both the neurodegeneration and proinflammatory responses in scrapie (Kim et al., 1999). Using NF?B knockout mice we can investigate the function of NF?B in scrapie infectivity and scrapie pathology.
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