Animal Research Alternatives and Animal Care : Present Results

The online protocol application used by UW-Madison requires researchers to demonstrate having considered alternatives to potentially painful procedure in a brief narrative statement. The narrative should describe how you have sought information on alternatives and why those alternative methods will or won't work for you.

EXAMPLES OF NARRATIVES:

Alternatives to Animal Use

• We looked for phantom model alternatives to perform as much of our algorithm development as possible. Phantom models are attractive both because of sparing the use of animals, but also because of the greater degree of control available to the experimenter. However live animal models are needed to replicate actual physiological and imaging conditions that will be encountered in patients.

• • Because certain questions can be addressed using cell lines or primary cell cultures, I use electronic sources and journals to keep track of new insights in these studies. However, there are a series of many events that must occur to disrupt hematopoiesis in the bone marrow (toxin distribution, metabolism, activation of bystander (stromal) cells, oxidative stress, release of mediators). I seek alternative methods to the use of animals that would reliably reproduce these interactions, but have not found any such in vitro system. As a result, some experiments need to be conducted in live animals.

Alternatives to Painful/Distressful Procedures

• The surgical procedure to access the epicardial surface of the heart would cause pain or distress. For our studies this procedure is always performed as a terminal surgery on anesthetized animals, however, to explore less painful alternatives a search using the keywords "thoracotomy [Species Z] refinement" and "sternotomy [Species Z] refinement" was performed on [date X] and revealed [N] citations. None of the cited sources revealed alternatives to our current approach that are compatible with the research goals.

• There are no alternatives to ovariectomy that achieve the experimental goal of chronic reduction of estrogens in the [Species Z]. There are alternative methods for administration of estradiol, but these require frequent handling and injections over long periods of time, and our proposed method of delivery via implanted pellets limits the period of stress to the time immediately surrounding the implantation surgery.

• To perform blood draws for the purpose of pre-infection MHC genotyping and to collect plasma, serum, and PBMC during the course of the experiment, a search was performed on [date X] with the keywords “blood draw AND [Species Z] AND (alternative OR refinement) AND (method or model)”.  This search revealed one citation that only identified alternative ways to cooperate with animals during this procedure.  Because our animals are infected with [Pathogen W] they need to be restrained or anesthetized during the procedure to protect the animal care staff, and this alternative is not suitable for our needs.

• Bronchoalveolar lavage may be performed in this study to evaluate induction of immune responses in the lung mucosa.  A search was performed on [date X] with the keywords “bronchoalveolar lavage AND [Species Z]  AND (alternative OR refinement) AND (method or model).”  This search revealed two citations, but neither were alternatives to the distressful procedure.

• EAE is a demyelinating disease of the central nervous system and it is used as an animal model of multiple sclerosis. EAE is induced by subcutaneously injecting an emulsion of myelin oligodendrocyte glycoprotein peptide (MOG), myelin basic protein (MBP) or PLP with complete Freund’s adjuvant (CFA). CFA is widely used in research to elicit robust immune responses. CFA is a water-in-oil emulsion containing heat-killed mycobacteria, and it is an effective means of potentiating cellular and humoral antibody responses to injected immunogens. However, if improperly or excessively used, CFA can cause undesirable side effects in animals. The major side effects may include skin ulcerations and draining sinuses with granulomas and even can induce an autoimmune disease, especially arthritis. Considering the adverse effects of CFA, the following guideline will be used to minimize or eliminate the major side effects of CFA: 1. Aseptic preparation of MOG: CFA emulsion and mice injection site; 2. The injection route is subcutaneous and only given one time; 3. The injection volume is no more than 0.1 ml per mouse; 4. After injection, the mice will be monitored carefully to avoid any painful or distress response occurring; any unusual response after injection of CFA will be reported to vet staff immediately. The Ribi adjuvant system is known to have a less intense inflammatory response than CFA. It is reported that Ribi Detox may induce a stronger cellular immune response, but less antibody response than CFA (J. Chang, Advanced drug delivery review, 1998:32). The used of Ribi adjuvant in the induction of EAE is not well demonstrated so far. We will consider it as an alternative if it is demonstrated as a successful and stable adjuvant in EAE induction in the future.

• We have checked ‘PubMed’ information monthly to see if our proposed work unnecessarily duplicates existing knowledge and concluded that little is known about this proposing study. Also we have investigated what other options – or alternatives – exist that could be applied to our own research. However live animal models are needed to replicate actual physiological response. I seek alternative methods to the use of animals that would reliably reproduce these interactions, but have not found any such in vitro system. As a result, this experiment needs to be conducted in live animals. The keywords used for this search were  ‘PGC-1’, ‘immobilization’ and ‘mitochondrial biogenesis’.

• In these studies, we are not injecting the animals with any drugs. We will subject rats to focal ischemia surgery under isoflurane anesthesia. There is no alternate focal ischemia procedure to the surgical procedure of transiently occluding the middle cerebral artery. We routinely search PUBMED for any new methods/procedures which are less painful or need less surgical manipulation and can be used to induce focal ischemia in rats and will adapt to the new procedures if available.

Avoiding Unnecessary Duplication

• A few publications exist describing the effects of ovariectomy and/or estradiol treatments for manipulating expression of GFAP in the [Species Z] , thus providing a rationale for our proposed studies.  However, none of these existing studies have tested whether hormonal regulation is important for GFAP in Huntington’s disease, or whether estrogen supplementation can be used as a therapeutic strategy.