Attachment C - Vertebrate Animal Scientific Review Instructions.pdf

Vertebrate Animal Scientific Review (VASR)

A. Vertebrate Animal Scientific Review (VASR)

If vertebrate animals are to be used, the following five points must be addressed completely by applicants in the VASR worksheet of their proposal:

1. Detailed description of the proposed use of the animals, including species, strains, ages, sex and number to be used

2. Justification of the use of animals, choice of species and numbers to be used, and proposer’s assessment of potential benefits and knowledge to be gained.

3. Information on the veterinary care of the animals

4. Description of procedures for ensuring discomfort, distress, pain and injury is minimized

5. Method of euthanasia and the reasons for its selection

Each of the five points must be addressed, for all performance sites, in the VASR worksheet.

The VASR worksheet will be reviewed by the scientific merit review panel and the proposal coded as either No Vertebrate Animals, No Concerns/Acceptable, or Concerns/Unacceptable. If coded as Unacceptable, NASA staff will work with the applicant to resolve concerns prior to award. Coding of the proposal as Acceptable or No Vertebrate Animals is required prior to award.

In order to be coded as “No vertebrate animals” the vertebrate tissue used in the study will be obtained from other sources (e.g., tissue repository, animals euthanized for an unrelated purpose). The source of the tissue should be included in the VASR to validate the coding as no vertebrate animals used. If vertebrate tissues are obtained through euthanasia for tissue harvest, the proposed research is coded as use of live vertebrate animals. The generation of custom antibodies is coded as use of live vertebrate animals.

A “performance site(s)” is defined as the institutions where procedures with animals will be performed. If the applicant institution is not the site where animal work will be performed, the performance site must be identified. If there is more than one performance site, the description of animal care and use at each site must be included and must address the five points.

Applicants should be aware that NASA may release information contained in funded proposals pursuant to a Freedom of Information Act request.

B. Instructions for Scientific Reviewers

These instructions are to assist Scientific Merit Review Panel (SMRP) members in the VASR review of the proposal.

Subsequent to evaluation of the VASR worksheet by a SMRP, all proposals are coded as either

No Vertebrate Animals, No Concerns/Acceptable, or Concerns/Unacceptable.

Coding as NO VERTEBRATE ANIMALS - If vertebrate tissue used in the study is obtained from other sources (e.g., tissue repository, animals euthanized for an unrelated purpose), the proposal is coded as no vertebrate animals used. The source of the tissue should be included in the VASR to validate the coding as no vertebrate animals used. If vertebrate tissues are obtained through euthanasia for tissue harvest, the proposed research is coded as use of live vertebrate animals. The generation of custom antibodies must be coded as use of live vertebrate animals.

Coding as NO CONCERNS/ACCEPTABLE or CONCERNS/UNACCEPTABLE - Coding is based on the review of the five required points for each of the performance sites.

Performance site(s): This is defined as the institutions where procedures with animals will be performed. If the applicant institution is not the site where animal work will be performed, the performance site must be identified. If there is more than one performance site, the description of animal care and use at each site must be included and must address the five points.

C. Detailed Instructions for Preparation of the VASR

These instructions are to assist applicants in preparing their VASR information.

Preparation of the VASR worksheet:

Typically, all of the required elements for the VASR can be addressed within 1-2 pages.

Point 1 - Description of animals and how they will be used A concise, complete description of the proposed procedures must be included in the VASR.

While additional details may be included in the Research Strategy, a coherent, albeit brief, description of the proposed use of the animals must be provided within the VASR. The description must include sufficient detail to allow evaluation of the procedures. Examples of the types of procedures that should be described include blood collection, surgical procedures, administration of substances, tumor induction and post-irradiation procedures. In describing the animals, investigators must provide the following information for each species and/or strain to be used:

• Species

• Strain

• Ages

• Sex

• Number of animals to be used

Point 2 - Justifications for use of animals Investigators must justify the use of animals in the proposed research. The justification must indicate why alternatives to animals (e.g., computer models, cell culture) cannot be used and should indicate the potential benefits and knowledge to be gained. In addressing this point, researchers are encouraged to consider means to replace, reduce and refine the use of animals.

Rationale for the choice of species must be provided. The rationale should indicate the advantages of the species chosen and why alternative species are not appropriate. If less highly evolved or simpler animal models are available, justification must be provided for using more advanced species. For example, the use of non-human primates (NHP), dogs or cats should be thoroughly justified. If NHP species are to be used, a comparison to other NHP species may be appropriate. If animals are in short supply, costly, or to be used in large numbers, provide an additional rationale for their selection and the number of animals used.

Estimates for the number of animals to be used should be as accurate as possible. Justification for the number of animals to be used should include considerations of animal availability, experimental success rate, inclusion of control groups and requirements for statistical significance; cite power calculations where appropriate.

Point 3 - Veterinary care Descriptions of veterinary care should indicate the availability of veterinarians or veterinary technicians. For example, the VASR might indicate the number of veterinarians and veterinary technicians associated with the applicant institution, and their proximity to the performance site(s). The frequency with which veterinary staff observe or monitor animals should be stated.

If survival surgeries are proposed, veterinary involvement or post-surgical monitoring should be described. For example, if animal use involves invasive approaches that might result in discomfort, distress or pain, the investigator should indicate if or when veterinary care is necessary. The indicators for veterinary intervention to alleviate discomfort, distress or pain should be described. The ways in which veterinary staff may intervene should be described.

Point 4 - Provisions to minimize discomfort, distress, pain and injury Procedures or circumstances that may result in more than momentary discomfort, distress, pain or injury should be identified. Methods to alleviate discomfort, distress or pain should be described. If pharmacological agents are used, the agent(s) should be specified by name or class.

Any additional (e.g., non-pharmaceutical) means to avoid discomfort, distress, pain or injury should be described briefly. The manner, circumstances and duration of all post-surgical provisions and care should be described. If special housing is necessary following surgery or manipulations, the VASR should describe these provisions, the duration and type of monitoring provided. If procedures (e.g., pharmacological or surgical) might lead to severe discomfort, distress, pain or injury, indicators for humane endpoints and euthanasia (e.g., severe infection, respiratory distress, failure to eat, tumor size) should be described. All of these issues are particularly important for survival surgeries. If multiple surgeries are proposed, these must be well justified and provisions to avoid any potential complications must be described. Describe how restraining devices will be used, if applicable.

Point 5 Euthanasia The method(s) of euthanasia must be described and must comply with the AVMA Guidelines on

Euthanasia. If the method(s) do not comply with AVMA recommendations, the rationale and scientific justification for use of the method(s) must be provided. The indicators for euthanasia

(i.e., termination of experiment or humane endpoints) should be stated. It is not sufficient to state simply that humane methods will be used, that are consistent with the recommendations of the

AVMA Guidelines on Euthanasia or the Institutional Animal Care and Use Committee (IACUC).

References Guidance in this document is based on NASA and PHS Policy, and federal requirements. The

NASA and PHS Policy incorporate the standards in the Guide for the Care and Use of

Laboratory Animals and require that euthanasia be conducted according to the AVMA Guidelines on Euthanasia. Additional background information and references are available on the Office of

Laboratory Animal Welfare website (http://olaw.nih.gov).

NASA Policy and Requirements http://nodis3.gsfc.nasa.gov/displayDir.cfm?t=NPD&c=8910&s=1B http://nodis3.gsfc.nasa.gov/displayDir.cfm?t=NPR&c=8910&s=1B

PHS Policy http://grants.nih.gov/grants/olaw/references/phspol.htm

Guide for the Care and Use of Laboratory Animals http://www.nap.edu/openbook.php?record_id=5140

AVMA Guidelines on Euthanasia http://www.avma.org/issues/animal_welfare/euthanasia.pdf

D. Worksheet to Assist in Addressing the Required Five Points of the VASR

Performance site(s):

The five points must be addressed for all performance sites.

__ If the applicant’s institution is not where animal work will be performed, are all collaborative performance site(s) identified?

__ If more than one performance site is planned, are descriptions of animal care and use for each site provided?

Point 1 - Describe the animals and their proposed use; address the following for all species to be used:

__ Species

__ Strains

__ Ages

__ Sex

__ Number of animals to be used

__ A concise, but complete, description of proposed procedures (i.e., sufficient information for evaluation)

Point 2 - Provide justifications for:

__ The use of animals

__ Choice of species

__ Number of animals to be used (cite power calculations, if appropriate) http://nodis3.gsfc.nasa.gov/displayDir.cfm?t=NPD&c=8910&s=1B http://nodis3.gsfc.nasa.gov/displayDir.cfm?t=NPR&c=8910&s=1B http://grants.nih.gov/grants/olaw/references/phspol.htm http://www.nap.edu/openbook.php?record_id=5140 http://www.avma.org/issues/animal_welfare/euthanasia.pdf

Point 3 - Provide a general description of veterinary care, including veterinary support that is specifically relevant to the proposed procedures. Indicate the following:

__ A brief account of veterinary staff and their availability

__ The regular schedule of monitoring of animals by veterinary staff

__ Any additional monitoring and veterinary support that may be required to ensure humane care, if relevant to the procedures proposed (e.g., post-surgical)

__ Indicators for veterinary intervention to alleviate discomfort, distress or pain, if relevant

Point 4 - Describe procedures to minimize discomfort, distress, pain and injury. Indicate the following:

__ Circumstances relevant to the proposed work, when animals may experience discomfort, distress, pain or injury

__ Procedures to alleviate discomfort, distress, pain or injury

__ Identify (by name or class) any tranquilizers, analgesics, anesthetics and other treatments

(e.g., antibiotics) and describe their use

__ Provisions for special care or housing that may be necessary after experimental procedures

__ Plans for post-surgical care, if survival surgeries are proposed

__ Indicators for humane experimental endpoints, if relevant

__ Describe the use of restraint devices, if relevant

Point 5 - Describe methods of euthanasia:

__ Describe the method(s) of euthanasia and rationale for selection of method(s)

__ Indicate if the method is consistent with AVMA Guidelines on Euthanasia

__ Provide a scientific justification for the choice of method if not AVMA recommended

E. Example of a complete VASR

(This VASR worksheet has been modified from the original. It addresses all five points concisely)

Vertebrate Animals Aims 1-3 will be addressed in vitro; Aim 4 will be addressed using a mouse model of ocular infection.

1. Female Balb/c mice will be used to determine if virions treated with enzyme can cause viral keratitis, and to test the in vivo efficacy of the test articles. The studies will require 700 mice, 4 to

6 weeks old. Based on prior experience, 70 groups, each including 10 mice will be required over five years to achieve adequate statistical power. Ocular infection is accomplished by scratching the cornea of anesthetized mice with a sterile needle and exposing the scarred portion of the cornea to inoculum. Test articles are applied directly to the scarified cornea as liquid or cream.

Following inoculation and recovery, mice are monitored for 30 days. With the mice under anesthesia, the eyes will be examined at intervals, microscopically, and are flushed with medium with 2% serum to determine viral titers. Thirty days post-infection, with the mice under deep anesthesia, the trigeminal ganglia are removed aseptically for viral assay, followed immediately by euthanasia.

2. The proposal is to study mechanisms for the prevention of ocular disease caused by viral infections, a leading cause of blindness in the US. Mice are needed for these experiments because no alternative in vitro model incorporates all elements of the mammalian ocular immune system; too little is known about this system for the development of computer simulations. Mice are a well accepted model for studying viral keratitis, assessing the virulence of viral strains and testing the efficacy of antivirals. Mice provide several advantages: a) The murine ocular immune system is similar enough to that of humans to allow extrapolation of the results; b) Their small size allows the use of smaller amounts of drugs for testing; c) The entire mouse genome is known and easily manipulated genetically, allowing extension of the work in future genetic studies. Female mice will be used due to compatibility issues. Balb/c mice will be used because they have intermediate resistance to infection. ABC-4 knockout and ABC-4 test-strains will be used. For the enzyme study, we will use 4 treatment groups: enzyme-1, enzyme-2, enzyme-3, and mock treated virus. We will also use different amounts of inoculum for each condition allowing a more accurate calculation as to the effect of the digestions on infectivity. For the test-article peptide study, we will use two formulations (one aqueous and one hydrophobic), test 4 different concentrations and also vary the treatment protocol. Two groups will receive a single dose of drug in each of the two formulations prior to the addition of virus to assess prophylactic activity. These groups will not receive any additional enzyme treatments. Two groups will be infected with virus and beginning 4 h post-infection, we will treat with each formulation and concentration 4 times daily for 7 days.

3. All mice are housed in the Animal Resources Center of the University. Animal housing rooms are under temperature and humidity control. The mice will not be subjected to water or food restrictions, and bedding material is placed in each cage. The facility is staffed by four full time veterinarians and six veterinary technicians; the veterinary staff is on site and a clinical veterinarian is available at all times. Animal care staff conducts routine husbandry procedures

(e.g., cage cleaning, feeding and watering) and checks animals daily to assess their condition.

Laboratory staff monitors mice when treatments are given, disease is scored or samples are collected for titering. The veterinary staff monitors mice in their home cages, weekly. If animals exhibit any indication of infection or distress, the veterinary staff confers with laboratory personnel to recommend appropriate antibiotics, analgesics or other pharmaceuticals. The veterinary staff may intervene or recommend euthanasia based on animal welfare concerns.

4. Mice will be anesthetized with isoflurane (3-5%) during the infection process, when treatments are administered and titer samples are collected. This eliminates the need for restraint devices and topical anesthetics that would interfere with the infection and disease process. For post-procedural pain relief, we will administer buprenorphine twice daily for the duration of the experiments (i.e., approximately two weeks post-inoculation). Death is not an endpoint for the studies; the Balb/c strain was chosen because of its resiliency and resistance to this particular virus. Our goal is to avoid severe infections leading to death. Though unlikely, if an animal reacts severely, it will be euthanized, based on humane indicators (e.g., failure to groom or feed).

These experiments involve no post-surgical survival animals.

5. All mice will be euthanized by cervical dislocation under isoflurane anesthesia. Isoflurane ensures that the mice are unconscious, while dislocation ensures quick death. This minimizes animal distress, is effective and efficient; it is consistent with the recommendations of the AVMA

Guidelines on Euthanasia.