TITLE: Intracranial injections of viral vectors in mouse striatum
AUTHORS: Shinil Raina, Simon Bossi, Stephanie J Cragg

[DESCRIPTION]
The following protocol outlines the steps necessary for intracranial injections of viral vectors in dorsal or ventral striatum in mice. These steps are generalisable for different viruses and injection sites, however care must be taken to optimise injection coordinates and virus titre. 

[STEPS]
SECTION: Pre-Surgery Preparation
2. Collect virus from frozen storage (-80°C freezer) and keep on ice. 
 

SECTION: Pre-Surgery Preparation
1. Check health of mice to undergo stereotaxic surgery: mice should look well-groomed and exhibit normal exploratory behaviour, have pink colouration to show good blood perfusion and oxygenation. 

DO NOT operate on mice that do not meet these standards or that appear abnormal. 
SECTION: Pre-Surgery Preparation
3. Prepare post-surgery recovery cages: include forage in the bottom of the cage, sizzle, cotton pads, wooden blocks, cylinder, pyramid house, long spout on drinking water bottles, wet mash food, metacam jelly.
SECTION: Pre-Surgery Preparation
4. Turn on the THERMACAGE recovery boxes; place some tissue in each chamber. 
SECTION: Pre-Surgery Preparation
5. Spray designated surgical area, anaesthetic trolley and stereotaxic frame with disinfectant spray, wipe down, repeat leave for 5 min, and wipe down once again. 
 



SECTION: Pre-Surgery Preparation
7. Ensure syringes, needles, sterile swabs, sterile 0.9% saline, drill, hair clippers, are available and laid out for surgery. You will also need isopropanol swabs and a black marker. Ensure that all surfaces to be touched during the surgery outside the sterile field are appropriately covered with either clingfilm or barrier film.
SECTION: Pre-Surgery Preparation
7.1. Ensure that a sufficient amount of isoflurane anaesthetic has been added to the vaporizer reservoir for surgery.
SECTION: Pre-Surgery Preparation
6. Ensure the surgical heating pad is on top of the stereotaxic frame.
 

SECTION: Pre-Surgery Preparation
7.2. You will need to prepare 4 syringes. These are the syringes for the Marcaine, Metacam, and two syringes for saline. The syringes should be filled slightly more than the amount needed for the average weight of the mouse (20 g) and placed briefly in the recovery chamber. One of the saline syringes should be filled all the way and placed on the sterile field to be used throughout the surgery.
 

SECTION: Pre-Surgery Preparation
7.3. Check oxygen tanks are full (at least one full one in reserve on trolley – often use 1.5 small cylinders per day).
SECTION: Pre-Surgery Preparation
8. Place microinjector arm onto stereotaxic frame if using microinjector and Hamilton syringe for injections (1 turn = 50 nL) 
8.1. Using the centrifuge tubes in the surgery area, make sure that the Hamilton syringe is functioning properly. This is done by taking up ethanol into the syringe and expelling it out. Repeat this three times. If working properly, the ethanol should be taken up and expelled quickly. 
 

8.2. Repeat the same process with the saline. 
 

9. Load the virus into the Hamilton syringe. 
9.1. Prepare the virus by spinning down the tube and cutting approximately halfway on the length of the tube. 
9.2. Secure the open tube using the Blu-tack on the ear bar. Tighten the ear bar to prevent movement.
9.3. Move the Hamilton syringe to the centre of the tube and lower carefully till it is inside the tube. Now using the microscope, lower it further until it is 90% of the way in the tube. 
9.4. Using a 200 nl/sec speed, load the appropriate amount of virus into the syringe. Withdraw the needle and move away from the surgical field. 
9.5. Secure the remaining virus using masking tape over the opening of the tube and put it back on ice.
10. Place autoclaved surgical instruments onto sterile drapes on top of designated surgical area.
10.1. Prepare Marcaine (1:9 Marcaine to saline ratio) for local analgesia. Take care to use insulin syringes that are individually packed and sterile.

10.2. Prepare Metacam (1:4 Metacam to saline ratio) in a centrifuge tube for analgesia.
10.3. Check that the following instruments are available on the drape: scalpel, needle-holder, two kinds of scissors (one for cutting the sutures and the second for the incision) and two forceps.
10.4. Prepare some diluted hibiscrub (1:10) and some sterile saline (in falcon tubes) and place next to surgical instruments. 
11. Turn on the surgical heating pad to allow it to heat up before the mouse is placed on it. 
13. Fill the Hamilton syringe with the desired substance.
SECTION: Anaesthetic Induction and Surgical Preparation
14. Collect mouse for surgery from holding room and record pre-operative weight and time.
SECTION: Anaesthetic Induction and Surgical Preparation
15. Connect anaesthetic induction chamber to the vaporizer and ensure flow is directed for induction. 
SECTION: Anaesthetic Induction and Surgical Preparation
15.1. Place paper towels into the induction chamber. 
SECTION: Anaesthetic Induction and Surgical Preparation
16.1. Once head is well shaven, quickly switch the anaesthetic flow from the induction chamber to the stereotaxic nose cone. Turn the isoflurane rate down to ~1.5-2 %.
SECTION: Anaesthetic Induction and Surgical Preparation
17. Gently move the tongue of the mouse out of the way and place the mouse into the nose cone. 
SECTION: Anaesthetic Induction and Surgical Preparation
17.1. Place mouse in ear bars: left hand ear bar fixed, place this into left ear canal, pull gently back, then insert right hand ear bar. 
SECTION: Anaesthetic Induction and Surgical Preparation
17.2. Once in place tighten right ear bar and position nose cone close to mouse and tighten nose cone.
SECTION: Anaesthetic Induction and Surgical Preparation
18. Place the heating pad under the mouse with a paper towel between. 
SECTION: Anaesthetic Induction and Surgical Preparation
17.3. It is extremely important that the head it tightly locked in place: you can check by applying an appropriate amount of pressure with your index finger. 
 

SECTION: Anaesthetic Induction and Surgical Preparation
18.1. Use bubble wrap and 96 well plates to lift mouse to the appropriate height (in line with the nose cone). 
SECTION: Anaesthetic Induction and Surgical Preparation
16. Once 1 breath/s breathing rate reached and righting reflex absent, shave mouse head in induction box using clippers. 
12. Place the probe under the drape to make sure the heating pad does not overheat the mouse when first put on the mat.
SECTION: Anaesthetic Induction and Surgical Preparation
18.2. Wrap the tail in bubble wrap for protection from the surgical heating pad.
SECTION: Anaesthetic Induction and Surgical Preparation
18.3. Turn on the surgical heating pad (set to 37 degrees Celsius).
SECTION: Anaesthetic Induction and Surgical Preparation
19. Cover mouse in surgical drape or cling film to maintain sterility. 
SECTION: Anaesthetic Induction and Surgical Preparation
19.1. Only place drape on once you are happy that the mouse is breathing at a steady level (no gasping), and no pinch reflex exists when the foot is pinched.
SECTION: Anaesthetic Induction and Surgical Preparation
19.2. If mouse is gasping or not breathing turn rate down/off immediately: mouse should recover quickly (~10-20 s) when removed from isoflurane. 
 

SECTION: Anaesthetic Induction and Surgical Preparation
20. Place eye lubricant onto eyes to prevent drying out. 
 

SECTION: Anaesthetic Induction and Surgical Preparation
21. Swab shaved head with ethanol swabs to get rid of any remanent fur.
SECTION: Anaesthetic Induction and Surgical Preparation
21.1. Clean starting from the centre, making your way to the edge of the shaved area.
 

SECTION: Anaesthetic Induction and Surgical Preparation
21.2. Once clean of hair, repeat the same process using diluted hibiscrub three times.
SECTION: Anaesthetic Induction and Surgical Preparation
22. Gently lift the skin using sterile iris curved forceps.


SECTION: Anaesthetic Induction and Surgical Preparation
22.2. Inject appropriate dosage of Metacam subcutaneously according to mouse weight.
SECTION: Anaesthetic Induction and Surgical Preparation
22.1. Inject diluted Marcaine (1:9) under scalp for pre-incision analgesia. 
 

SECTION: Anaesthetic Induction and Surgical Preparation
24. Prepare yourself for aseptic procedure: wash hands with Hibi scrub, dry hands with autoclaved paper towels and put on sterile surgical gloves.
SECTION: Anaesthetic Induction and Surgical Preparation
23. Inject saline subcutaneously.
SECTION: Surgery
25. Throughout surgery regularly check under drapes that mouse breathing rate is slow and steady and that depth of anaesthesia is appropriate. Also, regularly check mouse temperature on heat controller unit.

Make incision in-between ears towards back of skull (make just large enough so you can see both bregma and lambda and you are able to locate your drill site).
SECTION: Surgery
25.1. Keep the scalpel steady and make one uninterrupted incision. 

 

SECTION: Surgery
25.2. Use sterile swab to clear, as gently as possible, subcutaneous tissue to see lambda and bregma. 
 

SECTION: Surgery
26. Check DV position at bregma and lambda to ensure skull is flat (using Hamilton syringe needle under the microscope).
SECTION: Surgery
26.1. Ensure there is no more than a 0.1 mm difference between bregma and lambda DV position.
SECTION: Surgery
26.2. Move nose bar up or down to level skull (if required; by loosening uppermost screw and moving nose bar along pivot).
SECTION: Surgery
27. Once bregma and lambda are levelled on the DV plane, set the ML (x-coordinate) and AP (y-coordinate) of bregma to 0: then move to the desired injection site using your pre-selected coordinates. 
 

SECTION: Surgery
27.1. At the pre-selected coordinates, move the needle just above the skull. There should be enough room for the marker to make a dot under the needle. 
SECTION: Surgery
27.2. Take note of where on this dot the pre-selected coordinates are by moving the needle closer to the skull. 
SECTION: Surgery
28. Drill hole. 
 


SECTION: Surgery
28.1. Clear hole of bone debris with sterile saline and sterile swabs.
SECTION: Surgery
28.2. Pick through dura with sterile disposable needle tip.
SECTION: Surgery
28.3. Ensure that Hamilton syringe needle can access drill site, i.e. you drilled in the correct location. Keep the exposed brain tissue moist with saline.
SECTION: Surgery
29. Take the DV coordinates of the surface of the brain (you will see a slight change in the light refraction
when the needle makes contact with the brain surface). This point should be approximately halfway through the bevel of the needle. 
SECTION: Surgery
29.1. Ensure Hamilton is dispensing correctly. 
SECTION: Surgery
35. Remove mouse from ear bars. 
SECTION: Surgery
33. Suture incision with non-absorbable, monofilament suture (4-6 stitches usually).
SECTION: Surgery
32. Ensure that a sterile drape or clingfilm is covering the mouse and change into a new pair of sterile gloves.
SECTION: Surgery
30. Repeat steps 27, 28, and 29 if doing bilateral injections. 
SECTION: Surgery
29.4. If injection site is deep, retract 0.5 mm and wait 5 more min, then retract fully. 
SECTION: Surgery
29.3. Inject the substance slowly (no more than 200 nL/min) and wait 5 min to ensure appropriate diffusion. Retract syringe slowly (about 1 mm per min). 
SECTION: Surgery
29.2. Lower the Hamilton syringe to the DV site slowly (about 1 mm per min).
SECTION: Surgery
31. Check no debris under scalp, clear with sterile saline if necessary. 
SECTION: Surgery
34. Inject 0.5 mL of 0.9% sterile saline subcutaneously (0.5 mL per site). 
SECTION: Surgery
33.1. Optional: Add skin glue in between the individual sutures, no more than one drop should be needed for regular incisions. Use more if larger incisions were used. 
 

SECTION: Surgery
36. Turn off anaesthetic and oxygen. 
SECTION: Surgery
38. Record cage number mouse came from, date of birth of animal, genotype, tail markings, pre-op weight, post-op weight, time awake following surgery, all surgery co-ordinates, time injection took to complete,
volume of substance injected. Any other notable observations regarding health status of mouse during or after surgery or things which may eventually affect health of mouse should also be noted.


SECTION: Surgery
37. Weigh mouse and record post-op weight. 
SECTION: Surgery
38.1. If doing multiple surgeries, start the second surgery with a fresh set of tools.
SECTION: Surgery
38.2. Clean the used set of tools with a tootbrush and hibiscrub.
SECTION: Surgery
38.3. Soak the tools in Reprozyme (enzymatic cleaner) and bead steriliser prior to using in a surgery.
SECTION: Post-Operative Recovery
39. Allow mouse to recover in the THERMACAGE. 
SECTION: Post-Operative Recovery
39.1. Monitor mouse until awake and exploring/feeding/grooming. 
SECTION: Post-Operative Recovery
40. Move to surgical cage prepared earlier in Step 3.
SECTION: Post-Operative Recovery
40.1. Ensure metacam jelly is provided. Alternatively, metacam is available for subsequent daily administration (oral/s.c.), if not using the jelly method.
SECTION: Post-Operative Recovery
40.2. Place some old bedding into the new cage to make it familiar to mice and reduce the chance of fighting when rehoused. 
SECTION: Post-Operative Recovery
41. Monitor animals for 7 days postoperatively, including mouse weight and behaviour as per post-operative monitoring form. 
 

SECTION: Post-Operative Recovery
41.1. Fresh metacam jelly or subcutaneous injections should be provided for the first three days postoperatively (at a minimum, can be continued if required according to postoperative monitoring scoring). 
SECTION: Post-Operative Recovery
41.2. If mouse is losing weight and is below pre-surgery weight, place easily accessible forage in bottom of cage (banana chips etc.) and some jelly in a weigh boat in cage until mouse gains weight back.
SECTION: Post-Operative Recovery
41.3. If wound infected or animal lost more than 15% of its pre-surgery body weight for more than 48 hours, the animal should be culled.
SECTION: Post-Operative Recovery
42. Mice should be kept with littermates that have had surgery and not with those that have not. 
 

SECTION: Post-Operative Recovery
43. Mice should be monitored weekly, including weight and behaviour, until animal is culled for experiment.