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  • Placental Injection Protocol (Sterile Conditions) - Maternal Canal Embolization
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The protocol for placental microinjections has been adapted from the previously established cardiac-specific microinjection protocol (Rahman, 2021). Though similar in approach, placental injections are challenging with respect to the increased risk of bleeding and deformable nature of the organ. In order to ensure successful embolization and survival, the following requirements must be met; 

  1. Target of interest and needle tip must be maximally echogenic at the point of injection
  2. Placenta must be stabilized from the embryo side 
  3. If possible, must avoid advancing the needle through highly vascular regions of the placenta (identified by large amounts of flowing speckle)

Pre-surgery: Prepping the Mouse 

  • Print the following data record sheets 
  • Weigh and label mice (use sharpie to mark the tails) 
  • Calculate dosage of meloxicam to give 30 – 60 minutes prior to surgery (0.005 x Mouse weight)
    • Note the time the dose was given
  • Shave mice using Nair (while under anesthesia) and after you’ve removed the hair place mouse prone with head in nose cone to administer meloxicam subcutaneously
  • Allow mice to recover in their cage until time of surgery

Pre-surgery: Surgical Set-Up

  • Turn on ultrasound machine and create a new series
    • Use the General Imaging application and the Cardiac package
  • Turn on glass bead sterilizer
  • Turn on platform heater
  • Turn on heating pad and place sterile cage on top of it
  • Turn on hot plate and place water-filled flask with PBS-filled syringe inside
    • Place thermometer probe in the flask and maintain temperature at 37 degrees Celsius
  • Attach 3D motor plus 40 MHz transducer to the ultrasound rail system
  • Swap out taller platform stand for the shorter one
  • Attach the micro-injector controller
  • Set up non-sterile station in BSC
    • On a blue pad place:
      • Alcohol wipes
      • Small gauze squares
      • Iodine bottle
      • Cotton applicators
      • Ophthalmic gel
      • Masking tape
      • Ultrasound gel
    • Load needle
      • Take syringe with irradiated Shear Thinning Biomaterial (STB) and test product integrity by injecting a little bit out
        • If you can form structures with the product then it is still viable
        • Wipe tip of syringe needle so it is free of any excess product with an alcohol wipe
      • Place a petri dish on the stand and fill it with sterile PBS to the brim
      • Unscrew microinjection needle cap and remove the first rubber washer
      • Backfill the needle with STB, prime it and load the needle on the metal plunger
      • Place needle in the PBS solution and inject into the PBS dish, ensuring the product is injecting out
    • Use alcohol wipes first and then the virox wipes to sanitize the micro-injector unit and rail system and ultrasound platform (wait 5 mins)
    • Glad Press and Seal (GPS) every knob and area that your hands will be in contact with
    • Place 4 pieces of GPS aside for draping over the mouse
    • Put on sterile gloves
      • ***The remaining steps require help from the surgical assistant, every step from here on out needs to be done under sterile conditions
    • Place blue pad down
      • 18 large gauze squares
        • Lay down 3 gauze squares for where you’ll place your tools
      • Lay out surgical tools, silicon blockers, plasticine blocks, ultrasound gel, cotton applicators and sutures

Surgical Protocol 

  • Surgical assistant
    • Place mouse in induction chamber at 4% isoflurane 100% oxygen 
    • Apply ophthalmic gel
    • Transfer mouse to platform and maintain anesthetic at 2.5% isoflurane 100% oxygen
    • Secure paws, temperature probe with tape and apply ultrasound gel
    • Clean abdomen with alcohol wipes (x2), then iodine (applied to gauze, x1) and once more use an alcohol wipe
    • Drape mouse with GPS
  • Surgeon
    • Make an incision in the skin that is the size of the fetus for the given gestational age
    • Using scissors, separate the skin from the muscle layer by gently inserting the scissor tip (closed) and opening it 
    • Make a second incision along the linea alba
    • Using forceps, grasp the uterine tissue (between two conceptuses) and gently pull them through the incision
    • Using two blunted forceps, gently remove the entire uterus, count the number of embryos on each horn
    • Place the right horn back into abdomen using a cotton applicator
      • Do not touch the uterine artery or placenta, only push on the side with the fetus
    • Prop up approximately 5 embryos of the left horn on the keyhole-shaped silicon blocker and secure with plasticine
    • Secure a second moon shaped blocker on top the first
    • Apply ultrasound gel and lower the transducer
    • Visualize target
      • If you need more information, use the 3D motor
    • Advance needle into the field of view, ensure it is hyperechoic (in addition to the target) and inject product
    • Retract needle, remove ultrasound gel and apply 37 degree Celsius PBS to the placenta of the injected fetus
    • Place embryos back into the abdomen, suture the muscle layer and staple the skin
    • Place mouse in sterile cage to recover=

Post- surgery

  • Post-op day one change cage to a clean one with bedding
  • Check incision site for potential loss of staples or bleeding 
  • Look for signs of discomfort, administer meloxicam if needed

Collection Day

  • Administer hypoxyprobe-1 90 mins prior to collection under ultrasound guidance
  • Upon imaging, determine whether mouse survived
  • If so, collect doppler data from the ductus venosus, umbilical artery and the remaining canals
  • Sac dams, collect maternal liver first
  • Weigh placenta’s and fetuses, collect tails, drain fetuses in warm PBS
  • Store in fixative: 4% Paraformaldehyde (PFA) + 2mM ProHance 
    • Liver 2 days
    • Fetus 5 days
    • Placenta 1 day
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