Species:  Mouse. Animals were obtained from our own mouse colony housed in our campus’ animal facility at a weight ranging 17-28 grams. 

Anesthesia used:  Ketamine / Xylazine, Isoflurane as needed.

Preparation of the surgical area: The surgical area that was used was aseptically sterilized, had proper lighting and contained with the appropriate surgical equipment.  This room is designated as the surgery room for all rodent surgeries./procedures.  A sterile blue pad is laid on top of a heating pad.

Preparation of surgical instruments and supplies: All of the instruments and supplies (i.e. tools, gauze, staples, etc.) are sterilized by autoclave.  A homemade glass pipette that is needed is soaked in 70% ethanol.

Preparation of the animal: The animal was injected IP with Ketamine and maintained if necessary with isoflurane.   Ophthalmic ointment was then placed on the animal’s eyes to prevent them from drying out during surgery.  The animal is laid dorsal side down on top of the sterile blue pad.  The animal’s front limbs are taped down to the pad so that the chest is fully exposed.  70% ethanol is rubbed gently on the chest to moisten the hair so that it lays down.   The animal is now ready for the procedure.

Preparation for the surgeon: The surgeon wears a facemask, hair bonnet, surgical gown and clean, sterile foot covers.  Before beginning the surgery, the surgeon thoroughly washes her hands and puts on a pair of sterile gloves.  Once the surgeon is properly gowned and gloved, she places her sterile instruments on the sterile blue pad next to the microscope as well as sterile sutures and other necessary supplies. 

Monitoring: After the IP Ketamine injection to the animal, it is determined fully anesthetized when it lose its reflexes.  A nose cone contained isoflurane is used only if needed during the surgical procedure.  During preparation and surgery, the anesceptic depth is determined by the animal’s respiratory pattern and by pinching the animal’s foot for reflex response.  After the post-operative procedures, the animal is placed into a clean cage, which is placed on top of a heating pad at the appropriate temperature, lined with a blue pad.  The animal is monitored until it has awakened from the procedure and is moving around the cage normally.  This usually takes 30 minutes to one hour.  The animal is then observed on a daily basis by the surgeon until sacrificed.

Analgesia: The animal is given a subcutaneous injection of Banamine after the procedure is finished.  The animal is also given a subcutaneous injection of room temperature sterile saline directly after the surgery is finished, prior to waking up.

Operative description:  Once the animal is placed on the sterile pad and its limbs are taped down, the animal is ready for the procedure.  With a 26 gauge needle and a 1cc syringe loaded with your cells of interest and cell culture medium ( 100 ul total volume), inject the needle directly into  the chest between the animal’s sternum and third intercostals space, inserting the needle into the second intercostals space..  You are wanting to inject the cells into the left ventricle of the heart.  When you have inserted the needle into the left ventricle of the heart properly, blood will pump into your syringe needle like a pump.  When this happens you can begin to inject the cell solution, but at a very slow pace (approximately 30 seconds for total 100 ul).  Injecting too fast can kill the animal instantly due to the arteries being clogged.  Once the injection is complete, the needle needs to be taken directly straight out quickly.  After the injection is complete and successful, the animal is placed back into a clean cage, which is left onto the heating pad until the animal if fully recovered from the anesthesia and is walking around the cage.  The animal is monitored closely until has done so.