In the field of basic brain science research, intracranial stereotaxic injection is the most direct way to achieve precise injection in target brain regions, and has become an important part of most animal experiments such as injection of viruses, cells, protein molecules, drugs, labeled dye probes, etc. Combined with the matching optical fiber, it also enables optical stimulation or neuronal signal recording of the target brain region during, or after the injection. This method is widely adopted in animal models of human neurological diseases, advanced brain functions, emotions, cognition and other related studies.

Intracranial stereotaxic injection is usually applied in single administration and multiple administrations. During the experiment, the appropriate method can be selected according to the frequency of injection, the time of onset and the duration of action. In this paper, we will elaborate on these two methods in details, including the SOP and tips.

SOP of Cranial Single Administration

1 Instruments

Stereotaxic Apparatus
Laboratory Animal Anesthesia machine
Micro syringe pump
Micropipette puller

2 Process and procedure

2.1 Pre-surgery animal anesthesia

Turn on the animal anesthesia machine. Place the rat in the induction box to conduct induced anesthesia. Once the rat is under anesthesia, take it out from the induction box and transfer to the anesthesia mask at the stereotaxic instrument with a heating pad. Apply[XY1] eye ointment to their eyes and prepare the skin.

2.2 Fix the skull

2.2.1 Put the rat’s incisor into the hole of the mask and gently screw the nose clip.
2.2.2 Move the ear bar on one side to the appropriate position and screw it into place. Hold the rat’s head gently and align the position of the large occipital foramen on the same side of the head with the tip of the ear bar, and slowly push the other side of the ear bar to the large occipital foramen.
2.2.3 Adjust the position of the ear bars on two sides until the readings on left and right sides are the same.

2.3 Drill a hole on the skull

(2.3.1) Fix the micro drill on the brain stereotaxic instrument with the micro drill holder and slowly move the micro drill to touch the Bregma and set zero at the digital display. Slowly move the micro drill to touch the Lambda to record the value of DV. If the absolute value is less than 0.03mm, the skull can be regarded in the horizontal position; if greater than 0.03mm, the height of the adapter needs to be adjusted, and the positions of Bregma and Lambda need to be re-calibrated until the absolute value of Lambda DV is less than 0.03mm.

2.3.2 Move the cranial drill to the middle point of the Bregma and Lambda line and expand the left and right sides by 0.3mm. Touch the cranial surface gently, and record the craniofacial DV values at the left and right points respectively. If the absolute difference is less than 0.03mm, the skull can be regarded in the horizontal position; if greater than 0.03mm, the height of the two ear bars should be adjusted until the absolute difference is less than 0.03mm.

2.3.3 Slowly move the cranial drill to above the target site. Turn on the cranial drill, then slowly lower the cranial drill while observing the drill bit under the microscope until it reaches the dura.

2.4 Intracranial injection
2.4.1 Use the glass capillary to pull the glass capillary by referring to the parameters of injection program.
2.4.2 Use a filling needle to fill the glass capillary with mineral oil, and flick the glass capillary until the bubbles at the tip are discharged.
2.4.3 Loosen the chuck of the micropipette injection pump to gently push the glass capillary to the top, and then tighten the chuck.
2.4.4 Set parameters for emptying speed and emptying volume in the control unit and click to execute emptying.
2.4.5 Immerse the capillary tip in the solution to be injected. Set parameters for filling speed and filling volume in the control unit and click to perform filling.
2.4.6 Fix the micropipette injection pump on the operating arm. After resetting the capillary tip to zero, move the operating arm above the target site and slowly descend to the target brain region.
2.4.7 Set parameters for injection speed and volume in the control unit and click to execute injection.
2.4.8 After the injection, slowly move the micropipette injection pump upward until the tip of the glass capillary is removed from the brain tissue.

2.5 Suture
2.5.1 Suture the scalp with sterile suture, and then apply the erythromycin eye ointment to prevent wound inflammation.

2.6 Postsurgical care
2.6.1 Keep the rat warm for 24 h after the surgery. Inject penicillin or other antibiotics to prevent infection, and provide the rat with adequate feed and drinking water.

3 Tips

3.1 After the tip of the glass capillary reaches the target site, keep the capillary at the injection site for 1 min in order to balance the air pressure before injection.
3.2 In order to make the injection reagent fully absorbed by the brain tissue, stop the injection for 15-20 min after the injection, and then pull out the glass capillary.
3.3 In order to reduce the leakage of reagents on the moving trajectory of the glass capillary, it is suggest to control the needle inlet and outlet speed at 0.01mm/s.

SOP of Cranial Multiple Administration

1 Instruments

Stereotaxic Apparatus
Drug delivery cannula
Micro syringe pump

2 Process and procedure

2.1 Pre-surgery animal anesthesia
2.1.1 Turn on the animal anesthesia machine. Place the rat in the induction box to conduct induced anesthesia. Once the rat is under anesthesia, take it out from the induction box and transfer it to the anesthesia mask at the stereotaxic instrument with a heating pad. Apply eye ointment to their eyes and shave the skin on their head for preparation.

2.2 Fix the skull
2.2.1 Put the rat’s incisor into the hole of the mask and gently screw the nose clip.
2.2.2 Move the ear bar on one side to the appropriate position and screw it into place. Hold the rat’s head gently and align the position of the large occipital foramen on the same side of the head with the tip of the ear bar, and slowly push the other side of the ear bar to the large occipital foramen.
2.2.3 Adjust the position of the ear bars on two sides until the readings on left and right sides are the same.

2.3 Skull leveling
2.3.1 Fix the micro drill on the brain stereotaxic instrument with the micro drill holder and slowly move the micro drill to touch the Bregma and set zero at the digital display. Slowly move the micro drill to touch the Lambda to record the value of DV. If the absolute value is less than 0.03mm, the skull can be regarded in the horizontal position; if greater than 0.03mm, the height of the adapter needs to be adjusted, and the positions of Bregma and Lambda need to be re-calibrated until the absolute value of Lambda DV is less than 0.03mm.

2.3.2 Move the cranial drill to the middle point of the Bregma and Lambda line and expand the left and right sides by 0.3mm. Touch the cranial surface gently, and record the craniofacial DV values at the left and right points respectively. If the absolute difference is less than 0.03mm, the skull can be regarded in the horizontal position; if greater than 0.03mm, the height of the two ear bars should be adjusted until the absolute difference is less than 0.03mm.

2.4 Drill a hole on the skull
2.4.1 Slowly move the cranial drill to above the target site. Turn on the cranial drill, then slowly lower the cranial drill while observing the drill bit under the microscope until it reaches the dura mater cerebralis.
2.4.2 Slowly move the micro drill and drill two small holes in the front and back of the target brain region. Insert the sterile screws gently into the skull with screwdriver.

2.5 Drug deliver cannula implantation
2.5.1 Clamp the guide cannula and slowly move it to Bregma and reset the zero point. Then move the guide cannula to above the target site and slowly descend to the target area.
2.5.2 Apply appropriate amount of biological glue to the skull opening.
2.5.3 Prepare semi-viscous dental cement to fill between screw and guide cannula, layer by layer, zone by zone.
2.5.4 Screw the guide cannula cap tightly after the dental cement has completely solidified.

2.6 Drug delivery with cannula
2.6.1 Using the PE tube across the fixing screw and fill the PE tube and the injection needle with mineral oil. After the syringe is filled with mineral oil, use a glue gun to seal the connection between the PE tube and the syringe, and empty the mineral oil in the syringe.
2.6.2 Fix the syringe in the propulsion tank of the syringe pump. The syringe pump performs the suction procedure and inhales the drug from the tip of the injection cannula.
2.6.3 Remove the dummy cannula. Insert the injection cannula into the guide cannula
slowly, and tighten the fixing screw.
2.6.4 Let it rest for 1 min and enable the animals with injection tube adapt to the environment.
2.6.5 Set injection parameters of the injection pump and execute the injection procedure.
2.6.6 After the injection, stop the injection for 1-5 mins. Then remove the inner injection tube and tighten the catheter cap again. The injection is completed.

2.7 Postsurgical care
Keep the rat warm for 24 h after the surgery. Inject penicillin or other antibiotics to prevent infection, and provide the rat with adequate feed and drinking water.

3 Tips

3.1 When drilling holes at the position where cranial screw are embedded, special attention shall be paid in order not to drill through the skull and avoid brain tissue damage. Besides, the drilling location should be kept as far away as possible from the implanting area of the cannula to avoid the cranial screw blocking.
3.2 For bilateral implantation, implant the right single cannula first to prevent the spatial obstruction.
3.3 Apply the dental cement layer by layer to solidify the implantation.