Spring Program in Neurosciences 2011

References

1. Whishaw et. al, Analysis of Behaviour in Laboratory Rodents
2. Murphy, Two-Photon Imaging of Neuronal Structural Plasticity in Mice during and after Ischemia

Protocols

Middle Cerebral Artery Occlusion (from CCA)

1. Weigh the animal (ideally 280-330 g)
2. Anesthesize the animal (ideally inhalable anesthesia)
3. Clean the neck area (cut the hair and swab with iodine, followed by 70% ethanol)
4. Maintain the core temperature with the temperature feedback control heating pad; keep it between 37 ± 0.5 °C
5. Cut the skin along midline
6. Separate the subdermal and thyroid by forceps
7. Put a suture along the left side tissue, use a hemostat to hold it
8. Clear the common carotid artery (CCA)
9. Use a 5-0 suture to block the CCA, double knots (the lower the better; be sure no nerves inside)
10. Clear the bifurcation (there is a small artery just after the bifurcation, be sure to cauterize it)
11. Use a 5-0 suture to block the external carotid artery (ECA), double knots (the closer to the bifurcation the better)
12. Put the aneurysm clip to block the internal carotid artery (ICA)
13. Put a security 5-0 suture around CCA, make one loose knot
14. Cut a small hole on CCA
15. Put the 3-0 PDL-coated suture in the CCA, the round tip toward the bifurcation
16. Tighten the security suture
17. Take the aneurysm clip away
18. Push the 3-0 suture into the ICA, stop upon feeling mild resistance (if having difficulties putting the suture in, try putting the head of the animal at a different angle)
19. Start the timer
20. Make the security suture tighter
21. Close the skin temporally
22. Let the animal wake up, leave the animal under the heating lamp till it wakes up, wait for 1.5 hour
23. Measure the core temperature every 10 min
24. Do a behavioral test before re-anesthetize the animal
25. Reopen the skin
26. Take the suture out (just before the tip is out put the aneurysm clip around CCA)
27. Tighten the security suture, double knots
28. Close the skin
29. Measure the core temperature after 30 min stroke

Middle Cerebral Artery Occlusion (from ECA)

1. Weigh the animal (ideally 280-330 g)
2. Anesthesize the animal (ideally inhalable anesthesia)
3. Clean the neck area (cut the hair and swab with iodine, followed by 70% ethanol)
4. Maintain the core temperature with the temperature feedback control heating pad, make it between 37 ± 0.5 °C
5. Cut the skin along midline
6. Separate the subdermal and thyroid by forceps
7. Put a suture along the left side tissue, use a hemostat to hold it
8. Clear the common carotid artery (CCA)
9. Use a 5-0 suture to block the CCA, double knots (the lower the better; be sure no nerves inside)
10. Clear the bifurcation (there is a small artery just after the bifurcation, be sure to cauterize it)
11. Use a 5-0 suture to block the external carotid artery (ECA), double knots (the further away from the bifurcation the better); put a second 5-0 suture around ECA (even further away than the first one)
12. Use the heat coagulator to cauterize the ECA between the sutures
13. Put the aneurysm clip to block the internal carotid artery (ICA)
14. Flip the ECA over. Put a security 5-0 suture around ECA, make one loose knot
15. Cut a small hole on ECA
16. Put the 3-0 PDL-coated suture in the ECA, the round tip toward the bifurcation
17. Tight the security suture
18. Take the aneurysm clip away
19. Push the 3-0 suture into the ICA, stop upon feeling mild resistance (if having difficulties putting the suture in, try putting the head of the animal at a different angle)
20. Start the timer
21. Make the security suture tighter
22. Close the skin temporally
23. Let the animal wake up, leave the animal under the heating lamp till it wakes up, wait for 1.5 hour
24. Measure the core temperature every 10 min
25. Do a behavioral test before re-anesthetize the animal
26. Reopen the skin
27. Take the suture out (just before the tip is out put the aneurysm clip around CCA)
28. Tighten the security suture, double knots
29. Close the skin
30. Measure the core temperature after 30 min stroke

Photothrombosis Procedure for inducing Stroke

1. anesthetize mouse (30 grams) with an i.p. injection of 0.15 ml ketamine/xylazine solution
2. shave fur off the top of the head, and place ointment over eyes to prevent them from drying out
3. place mouse into stereotaxic apparatus, ensure that the head is snugly held by ear bars
4. ensure body temperature is properly maintained (36-37 °C) using heated blanket, rectal thermoprobe coupled to temperature regulator
5. before operating, make sure you have adequate anesthesia. Therefore test for loss of reflexes (tail pinch, eye blink)
6. using a scalpel, make a 1 cm incision along the midline of the scalp
7. retract the scalp using hemostats to expose the skull
8. use cotton tips to wipe off any fascia or tissue on surface of the skull, especially around the area where you want to induce a stroke (skull must be completely dry)
9. give mouse an i.p. injection of rose Bengal solution (100 mg/kg). Therefore if 30 gram mouse, give 0.3 ml of rose Bengal at a concentration of 10 mg/ml.
10. after the injection wait 2 minutes before turning on laser
11. irradiate particular part of the skull (where you want to cause a stroke directly underneath) with a 12 mW laser that is set at 532 nm (green) wavelength. The laser should be about 2-4 cm above the skull, typically with a diameter of 1-2 mm.
12. shine the laser for 15 minutes
13. suture up the scalp with 5.0 silk
14. monitor breathing, body temperature and general behaviour post-operatively to ensure successful recovery