TITLE: Surgical window preparation to visualize the trigeminal ganglion in an anesthetized mouse
AUTHORS: Elizabeth A. Ronan, Deanna N Cannizzaro, Joshua J. Emrick

[DESCRIPTION]
The trigeminal ganglion houses somatosensory neurons that innervate peripheral structures throughout the head and face. This protocol describes step-by-step instruction to perform a non-survival surgery for visualizing the ganglia. We apply this procedure to record calcium responses from the mouse trigeminal sensory neurons in vivo.

[GUIDELINES]
Animal subjects research was approved by the University of Michigan Institutional Animal Care and Use Committee. (IACUC protocol # PRO00010546).

[STEPS]
SECTION: Positioning the mouse in the stereotax
11. Induce anesthesia using isofluorane according to your approved animal protocol.
 

 



SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
18. Hold skin up with curved forceps. Cut and remove skin above the skull and carefully remove overlying connective tissue (scalp).
 

 

 

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
19. Use curved forceps to displace residual thin membrane from the top of skull, so the skull is clear of any tissue.

 

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
21. Use a small dental bur (#35 carbide bur) and gentle cutting to create a shallow outline on the skull in a broad pear shape (as shown in D). Start laterally and cut the medial parts in front and back last because the midline contains large blood vessels. Irrigate any bleeding with warmed 1X PBS and suction with the suction pipet.
 
  
 


SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
22. Gently lift off detached skull with curved forceps. Assume that bleeding will now begin.


 

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
24. Begin to suction out the cortex with the large suction pipet, starting anteriorly and traveling around the edge of the window on the left and right sides. Blood may pool as portions of brain are removed. Continue to irrigate, and suction to remove pooling blood. Use small spring scissors to cut any remaining dura (possibly near anterior midline). 
 


 

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
30. Fill the skull with 37°C 1X PBS and reduce isofluorane an additional 1/4th of the starting concentration (e.g., if starting at 2%, the isofluorane should be reduced to 1.5% during the procedure, and 1% once the brain is removed).
SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
33. Within the skull, you should now observe a blood clot with a gelatin-like consistency. Begin removing the clot by suctioning from the sides of the skull, and dragging the clot across the TG to uncover it. Remove all of the clot, but do not touch the TGs with the suction pipet. 
 

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
34. TGs should now be fully exposed. Well done. If some bleeding continues, fill the skull with PBS and suction away. Repeat 2x and bleeding will typically subside.

If you encounter excessive bleeding return to   

SECTION: Positioning the mouse in the stereotax
12. Remove mouse from induction chamber and change the anesthesia flow to the nose cone (2% isofluorane, flow rate 125mL/min). Parameters may need to be adjusted depending on mouse size and breathing.
 

 

 


SECTION: Positioning the mouse in the stereotax
13. Position incisors (through the nose cone) above the palate bar hole. Tighten the top clamp down onto the mouse snout with nose cone to prevent nose cone displacement and secure the position of the mouth.
 

SECTION: Suction Pipets Fabrication
3. Clean the workstation near a bunsen burner, removing any flammable items from the area. Ensure no loose clothing, body parts, or hair would fall into the flame.


 

  

SECTION: Suction Pipets Fabrication
4. Turn on the bunsen burner and ignite. Hold a single Pasteur pipet over the open flame at the tapered portion of the glass. 

  
SECTION: Suction Pipets Fabrication
5. While steadily holding the Pasteur pipet, gently torque each end in opposing directions while applying inward pressure.
SECTION: Suction Pipets Fabrication
6. As soon as the glass begins to become malleable, remove the Pasteur pipet from the flame and immediately pull each end outward in opposing directions until glass capillary extends to the desired width. Allow the pulled pipet to briefly cool, taking care not to touch the hot portion of the glass.
 

SECTION: Suction Pipets Fabrication
7. Gently hold the pulled pipet at the base parallel to the bench, and submerge the elongated portion in the top of the flame at a 45-90° (based on preference) so that the glass again becomes malleable and the end drops to a ~90 ° angle from the base.  

SECTION: Suction Pipets Fabrication
8. Set the pipet on the bench, with the new pulled suction end resting on the bench surface. Use a folded piece of    (grain side out) to create a knick in the angled end at the desired end length of your vacuum tip.
 


SECTION: Suction Pipets Fabrication
9. Once knicked, apply tension from the tip of the knicked end until the glass breaks neatly to the desired vacuum length.


 
 
  

SECTION: Suction Pipets Fabrication
8.1. For larger vacuums, you may need to knick both sides to create a weak point.
SECTION: Stereotaxic Surgical Apparatus Assembly
1. Any stereotax that stabilizes the skull and enables access to the mouse would be sufficient.
We assembled a custom stereotax from the following parts:
        
 
The palate bar was obtained from Harvard Apparatus hardware: https://www.harvardapparatus.com/standard-u-frame-stereotaxic-instrument-for-mouse-and-neonatal-rat.html

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
17. Trim whiskers and wet fur on top of the head with PBS.



SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
23. Upon removing the skull cap, reduce isofluorane concentration by 1/4th of the total percentage (e.g., if at 2%, lower to 1.5%). 
SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
25. Once the cortex within the window is removed, the underlying hippocampus will be fully exposed. At this point, suction away remaining brain at the anterior aspect of the window. Do not cross the midline. Continue to use a rapid blotting motion and regular irrigation. 
SECTION: Stereotaxic Surgical Apparatus Assembly
2.   

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
26. As you remove deeper layers, avoid the optic chiasm. Continue focus on removing the anterolateral regions until the trigeminal ganglia (TG) are only visible at the rostral portion. 
 


SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
27. Once the TGs are visible, switch to a medium suction pipet (1 mm) and gently suction at the midline to remove brain tissue above the optic chiasm.  Ideally, the optic chiasm will be fully exposed and intact, with the brain above it removed.
 

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
28. Before continuing to exposing the caudal portions of the TGs, suction the portions above them to remove additional brain without exposing them yet. Avoid the brain at the caudal midline which lies over the brainstem. 
 
 
SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
29. Once the majority of the brain is removed, except for a thin layer directly covering the ventral portions of the TGs, remove the remaining brain obstructing view of the TGs.
 

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
32. Optional step:
Depending on the stimulation being performed during imaging, additional stabilization of the head may be needed to prevent shifting of the focal plane. We are happy to discuss additional methods for immobilization.

   

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
31. Now allow large blood clot to form over TG for 3-5 minutes. 

Optional step: While waiting, we recommend replenishing fluids by giving the mouse an IP injection of sterile saline heated to 37°C. (For males inject 0.5mL, for females, 0.25mL). 
  

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
36. TG are exposed and are now ready for imaging. 
 

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
16. Warm 1X PBS to 37 °C using a standard laboratory hot plate.
  


 



We use a Pasteur pipet with a 1mL bulb to pipet PBS during the procedure

  
  
SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
35. Optional step:

If a clot cannot be removed from the TG surface, flow a shallow layer of PBS above the TG and use the large end of fine paper point saturated in PBS to mechanically dislodge the clot on the surface of the TG. We do not recommend physically contacting the TGs except as a last resort (i.e., here)

  

SECTION: Procedure Overview
10.  
  

SECTION: Bilateral Hemispherectomy and Exposure of Trigeminal Ganglia
20. Connect prepared large (1.5 mm) suction pipet   to vacuum tubing and vacuum trap. Turn on the vacuum valve.  

 

SECTION: Positioning the mouse in the stereotax
14. Position ear bars in the ear canals so that head is immobilized with the top of the head as flat as possible. A line through bregma to lambda should also be parallel to the ground.

 

SECTION: Positioning the mouse in the stereotax
15. Wet eyes with artificial tears to prevent drying.