91´ÎÔª

Footpad Inoculation and Immunization in Rodents

Number: IACUC-POL-036 

Responsible Office: Office of Research and Creative Scholarship (ORCS) 

Applies to: Principal Investigators Conducting Animal Research 

1. Purpose 

Thie purpose of this policy provides detailed guidance for performing footpad inoculation and immunization in mice for research purposes. The procedure is used for evaluating immune responses, vaccine efficacy, and localized inflammatory reactions. This policy and corresponding SOP ensures the procedure is carried out consistently, humanely, and in compliance with institutional animal care and use policies, the Guide for the Care and Use of Laboratory Animals, and relevant regulatory standards. 

2. Introduction 

Footpad injection is a classic method of inoculation and immunization. This method produces significant pain and distress in animals and may result in infection and excessive inflammation at the injection site, resulting in unrelieved pain and progressive debilitation. The use of footpad injection is strongly discouraged. Rodent forefeet are used for handling food, grooming, and copulation; hind feet are considered major weight-bearing structures.  

The animal welfare issues surrounding this injection method have been a subject of study for more than 30 years. The animal research field has progressively discouraged and decreased the use of this method over time as knowledge and standards have improved. The NIH Animal Research Advisory Committee (ARAC) Guidelines discuss footpad injections, stating that “whenever possible, the least invasive methodology required to accomplish an experimental goal should be utilized, and more invasive injection routes should be avoided unless scientifically justified.”  

UM IACUC Policy: Consistent with the NIH guidelines and the consensus of the research community, footpad injections are not permitted for any purpose, aside from a model of pain, unless strong scientific evidence is provided to the IACUC that this is the only effective method, not just the superior method, to achieve a specific research objective.  This evidence must be provided as part of the IACUC protocol. 

3. Procedures 

1. The preferred alternative methodology to footpad inoculation/injection is hock injection. Numerous studies in recent years have shown effective alternatives to footpad injections, which produce equivalent or comparable immunization, immune response, lymphatic drainage, and tumor inoculation. The principal recommended alternative to footpad injection for immunological studies is subcutaneous injection in the hock (the lateral tarsal region just above the ankle). The hock is a non-weight-bearing structure that drains to the same lymph nodes as the footpad. Additionally, the subcutaneous space is significantly larger and will accommodate larger injection volumes than the footpad with reduced reflux after injection.

1. 1. Hock injections have been shown to produce equivalent immune responses to footpad injections in numerous inbred mouse strains using different antigens and heat-killed bacteria.  
   2. Hock injections produce significant and effective uptake of Evan’s blue dye, PET, and fluorescent imaging agents in popliteal, sciatic, and inguinal lymph nodes 
   3. Hock injections have also been shown to induce comparable adenocarcinomas to the footpad injection when inoculated with MatLyLu tumor cells.  
   4. Volume to be administered into the hock region is limited to 100 μL in adult mice and 200 μL in adult rats.  
      
      
2. If the IACUC approves the use of footpad injections based on strong scientific justification that footpad injection is the only effective method to achieve a specific research objective, the investigator must follow the guidelines outlined in the NIH ARAC Guidelines.  
   1. Only one hind foot may be used
   2. Anesthesia is strongly recommended as a method of restraint. A restraint tube may be used in unanesthetized rodents; however, the foot will retract during injection, making it more technically challenging. 
   3. The hind foot to be injected should be thoroughly cleaned with disinfectant soap or 70% alcohol. 
   4. Sterile syringes and needles (25 gauge or smaller) should be used. A new needle should be used for each animal. The maximum injection volume is 50 μL for mice and 100 μL for rats. 
   5. If an adjuvant is used, volume must be limited to the minimum effective volume 
   6. With the bevel facing the skin, push the needle into the skin past the bevel before injecting. Withdraw the needle slowly while keeping pressure on the injection site. Continue firm pressure for several seconds following injection, then check for leakage of injected material. If leakage occurs, reapply pressure until it stops. 
   7. If anesthesia has been used, place the animal in a recovery cage with a heat source until the animal can right itself. When the animal is mobile and alert, it can be returned to its home cage. 
   8. Animals should be housed with deep, soft bedding following injection. 
3. Post Injection Monitoring and Care: 
   1. Animals receiving footpad injections must be monitored daily (including weekends and holidays). 
      • Any signs of pain or distress must be reported immediately to the AV. 
   2. Daily monitoring documentation (including weekends and holidays) must be left in the housing room for AV and LAR staff oversight. 
   3. Injectable analgesia must be administered for at least one day following footpad injection. Scientific justification must be provided in the approved AUP if analgesics cannot be given. 
   4. Animals should be monitored daily for 4 weeks or until all lesions have healed and there is no sign of pain or discomfort.
   5. Wet chow and/or dry chow in a dish on the floor of the cage must be provided to allow ease of access to food if the animals are unable to stand comfortably on their hind feet. 
   6. Volume to be injected into the hind limb footpad is strictly limited to 50 μL in adult mice and in 100 μL adult rats
   7. May not be used for routine immunizations 
   8. Footpad injections of any kind are prohibited in rabbits because they lack a true footpad 
4. Further guidelines from the IACUC are:  
   1. If multiple injections are required, the maximum amount of time possible between injections (two weeks is recommended as a minimum) between injections and must be clearly stated in the Animal Use Protocol (AUP) 
   2. Complete Freund’s Adjuvant (CFA) is not permitted to be given in the footpad under any circumstances 
   3. Objective humane endpoints must be clearly described in the approved AUP 

4. Reference 

1. Apostolico et al. 2016. Adjuvants: Classification, Modus Operandi, and Licensing. Journal of immunology research. 
2. Kamala et al. 2007. Hock immunization: a humane alternative to mouse footpad injections. Journal of immunological methods, 328 (1-2), 204-214. 
3. Barclay et al. 1988. The Disturbance Index: A Behavioral Method of Assessing the Severity of Common Laboratory Procedures on Rodents. Potters Bar: UFAW, p 36. 
4. NIH ARAC Guidelines, Guidelines for the Use of Adjuvants in Research.  
5. Morton et al. Refining Procedures for the Administration of Substances. 2001. Laboratory Animals, 35(1), 1-41. 
6. Wang et al. 2015. In Vivo Albumin Labeling and Lymphatic Imaging. Proceedings of the National Academy of Sciences. 
7. Richter et al. 2018. Comparison of Hock and Footpad Injection as a Prostate Adenocarcinoma Model in Rats. BMC Vet Res, 14, 327.  

5. Review, Approval and Version History 

Review and approval version history for this policy

Version	Date	Description of Changes	Approved By
1.0	January 13, 2026	Initial policy creation	IACUC Committee