Table 1.

Troubleshooting Guide for Parabiosis Surgery

Problem	Possible cause	Solution
High mortality rate after surgery	The most common complications after surgery are dehydration, malnutrition, hypothermia, dehiscence, pain, and infections. Evidence of these conditions are noted by excessive weight loss (>5%), lethargy, unequal growth of one mouse open wounds, and possible chewing or licking at clips and incision sites.	These complications can be treated effectively if found at early phase, therefore, it is important to perform health monitoring of the mice during recovery from parabiosis surgery. If dehydration is observed, fluids should be supplemented by injecting each mouse with 500μL-1 ml of sterile body temperature saline (subcutaneous injection) twice daily for 3–7 days (volume based on severity of dehydration). If weight loss or decreasing body condition is observed, place cage on heating pad for supportive care and provide additional options for supplemental food including wet chow and diet gels. To prevent infection, house mice in autoclaved cages after surgery, provide sterile water, and the water can be supplemented with antibiotics for prophylactic treatment. Multi-modal analgesia may be considered if signs of pain are noted. In addition to buprenorphine and the local analgesic, NSAIDS such as carprofen or meloxicam may be added to the analgesic regimen. Moreover, failure to accurately match weights of mice can be a big risk factor. The weight difference should be less than 10% difference in weight at the beginning of cohousing. The mice should be weighed again before the surgery. The weight difference should still be less than 10% difference in weight.
Poor induction of blood sharing	The common cause of poor blood sharing is poor wound healing and low angiogenesis due to inappropriate close of incisions.	While training on closure techniques is imperative prior to starting planned surgery, new surgeons may benefit from a surgical assistant at the time of closure. One person can hold the skin flaps from the dorsal sides of both mice up and together using two curved end forceps. In the meanwhile, another person can apply clips to close incisions. To achieve better angiogenesis, it is very important to have the outer edge of skin flaps exposed and face to the clips at this step. It is important to prevent the skin or remaining hair from clipping into the body during this procedure. Make sure the inside of the skin flaps is in touch when applying clips. It is also helpful to pull skins up when holding the margins together for clipping, this can improve clip placement and avoid inclusion of deeper tissue structures.
Lack of mobility of forelimb and/or hindlimb	This is usually due to inappropriate clipping of tissues (e.g., muscle).	Take off clips that impinge forelimb or hindlimb, reapply new clips. In cases where clips continue to impede movement, tension relieving sutures may be applied.