Before starting the surgery, mentally run through all the steps. Map and plan out the implant site and coordinates. Autoclave the instruments at 250 F for 15 minutes with about 4L of water. Start to fill out surgery sheet. Be sure to check:
Oxygen tank level
Isoflurane level
All necessary equipment is autoclaved
Bead sterilizer is turned on
Heating pad is turned on
Meloxicam, atropine, and lactated Ringer's solution are available with injection needles pulled out
Charcoal filters are below maximum weight
Grab rat. If you're using a rat retired from another project, double check the rat log / experimenter to make sure it is available.
Clean out the anesthetization chamber with chlorhexidine. Ethanol will damage the chamber.
Turn on the oxygen tank and set the flow rate to XXX. Make sure oxygen is running to the anesthetization chamber.
Place the rat in the chamber and latch the lid. Turn on the iso and set it to about 4% concentration. Leave the rat in the chamber for about 5 minutes or until breathing slows into spurts. (The gap between breaths should be about 2x longer than the breath itself.)
Turn off the iso and pull the rat out of the chamber. Get its weight and record in on the surgery sheet.
Fasten the rat in the nose cone making sure its front teeth are latched onto the mouth bar. Make sure the oxygen switch is turned to flow to the nose cone. Turn the iso back on to about 2.5-3% adjusting as needed. Always keep track of the rat's breathing.
Measure out the medications. The two medications used are
Meloxicam at 1 mg/kg -- a pain reliever / analgesic
Atropine at 1 mg/kg -- reduces mucus and saliva production
A solution of each medication should already be mixed. The concentration of each medication was set such that the appropriate dose can be measured as 1 mL/kg of the rat. (For example, if a rat weighs 500g, he will need .5 mL of meloxicam and .5 mL of atropine.) To measure out the meds, first pull out the syringe to the volume you wish to administer such that the volume of liquid in the solution container is replaced with an equal amount of air. (This is to avoid decreasing the pressure within the container.) Hold the bottle upside-down, push the needle up through the rubber top, and empty the air into the container. Pull some liquid into the syringe and quickly push it out to remove air bubbles from the top of the syringe (repeat if necessary). Fill the syringe with the appropriate amount of liquid and pull out of the container.
Administer each medication subcutaneously. To administer the medication, pinch the rat's skin behind the neck and pull up to form a "tent". The purpose of pulling up a tent of skin is to make sure the needle is placed underneath only the skin and not puncturing any muscles or organs. Flatten out the front of the tent with your finger -- this will be the application site. Push the needle into the tent (it should be parallel to the rat's body, NOT going in towards muscles and organs) puncturing the skin, and administer the medication at a moderate speed. Do this separately for each medication.
Shave the rat's head over a garbage can. (Note: the Oster razor blade work best at the edges.) Hold the rat around its rib cage and pull back the skin on his neck such that the skin on its head is taut (this will allow for a closer shave). Shave the head generously making sure to get around his ears, around eyes, down the nose between the eyes, and down the back of his neck. IMPORTANT: Avoid shaving any whiskers. Whiskers are very important for rat behavior and navigation -- shaving off a whisker is like chopping off a finger. The whiskers on the nose are the most important; the ones around the eyes aren't as critical. However, you can't shave too much fur -- think of the rat's grooming pattern. You don't want it to groom junk into the wound while its healing. If the rat begins to wake up while shaving, put it back in the nose cone and wait until it goes back down. If you're worried about the rat waking too quickly out of the nose cone, you can finish shaving his head on the table while keeping the rat in the nose cone. There is nothing wrong with this approach, it just makes the surgery table messier as now there will be fur on the table.
Put the rat back in the nose cone and apply Altalube generously to the eyes. Don't be afraid to lightly touch the eyes with the applicator or get the gel underneath eyelids: this is what projects his eyes from debris and keeps them hydrated.
Place the rat's head firmly between the ear bars. You're aiming to pin the ear bars underneath a ridge of bone (ADD PICTURE). If the ear bars are properly placed, the ears should lay flat on the bars. TIP: Fasten one ear bar to about 8-8.5 mm to hold it in place. Roughly align the fastened ear bar to hold the rat's skull in place; with the skull resting against the fastened ear bar, carefully align the loose bar into place and tighten. With the second bar aligned, realign the first bar to make sure it is also properly placed. With both bars aligned, simultaneously adjust the horizontal position of each bar such that they are equidistant (usually around 8-8.5 mm). After both ear bars are in place, press firmly onto the skull to make sure it stays in place. Don't be afraid to exert quite a bit of pressure: if the skull is going to become dislodged, it's better to happen now than when you're drilling into the skull later.
Clean the shaved area with chorhexidine scrub and ethanol solution (both should already be made up in the surgery suite). Using one or two cotton swabs, dip the cotton end/s into ONE of the solutions (do not mix), and begin cleaning at the center of the shaved area. Rub the cotton tips out from the center in a circular pattern, pushing debris away from the center out to the edges of the shaved area. Alternate between the two solutions and repeat this process about three times for each solution, using new cotton swabs each time, discarding used ones.
Lay out your sterile field onto the table and unpack all sterile equipment onto the field including the scalpel blade. Put on sterile gloves; avoid touching the sterile side of the gloves with your hands or other unsterile equipment. With your sterile gloves on, organize your sterilized surgery equipment and make sure everything is accounted for. If there is anything you forgot to sterilize, you can stick it in the bead sterilizer for about 15 seconds. Be sure to pour saline from a new container into one of the sterilized glass dishes. If you do this yourself, you may need to change your sterile gloves. Otherwise, have an assistant pour the saline for you.
Use the toe pinch tweezers to pinch the skin on the bottom of the rat's foot (you can also use hemostats). Pinch hard and check the leg for any muscle reflexes. Don't be afraid of hurting the rat -- any pain from a skin pinch would be dwarfed by the pain of the scalpel blade, so make sure now that the rat is fully anesthetized. Increase iso if you see any flinching. Repeat this step throughout the surgery along with checking the rat's breathing. If the rat ever flinches from the skin pinch or breathing speeds up, these are signs that the iso needs to be turned up.
Assemble the scalpel blade, making sure the blade clicks firmly in place. There are two methods to make the incision:
Randalyn's method: Press cotton swab on rat's head above incision site. Pierce skin with scalpel blade underneath the cotton tip. Don't be afraid to press hard -- you want to make sure you fully penetrate the skin and periosteum, and any nicks made to the skull are negligible (we'll be drilling into it later anyway). Holding the scalpel blade in place, use the cotton swab to pull up the skin to make the incision. After fully pulling up the skin, release the cotton swab and allow the skin to settle back in place. If you need to extend the incision further, move the scalpel blade further down the skull in line with the first incision, and pull up the skin again with the cotton swab. Aim for the incision to end before the neck muscles, but have it large enough such that enough of the skull is exposed for the implant site and anchor screws. Try to keep the cut in a smooth, straight line; avoid any jagged edges.
Ehren's method: Using a cotton swab, pull the skin taut on the top of the head towards the back of the neck. Holding the skin taut, pierce the skin towards the front of the skull with the sharp edge of the blade pointing towards the tail of the rat (the direction you pulled the skin). Once the blade has pierced through the skin, release the pulled skin while holding the blade in place. The blade will cut through as the skin settles. If the incision does not expose enough of the back of the skull, repeat this process except in the other direction. In other words, pull the skin taut towards the nose of the rat with a cotton swab, and pierce the skin over the back of the skull with the sharp edge pointing towards the nose of the rat. Be sure to align the placement of the blade such that it aligns directly with the first incision to make the final incision as straight as possible with no jagged edges.
Use cotton swabs to clean the area and push back the periosteum underneath the skin to expose the skull. Push the membrane back just over the bone ridges outlining the top of the skull. Wipe up as much blood as you can, replacing cotton swabs once they become too saturated. The bregma and lambda landmarks on the skull should be clearly visible.
After the periosteum has been sufficiently pulled back, use the hemostats to hold the incision open. Four hemostats should have been autoclaved for this purpose as they will pull back the four "corners" of the incision. Do not clamp on to the skin. Clamping onto the skin will cause the skin to die and necessitate cutting it off -- this leads to large jagged edges in the incision which will lengthen recovery and increase risk of infection. Each hemostat will clamp on to the periosteum to hold open the incision. To get a good grip on the periosteum, first hold back the skin with a tweezers of extra pair of hemostats and expose the underlying periosteum. Latch the hemostats onto a good portion of the periosteum, and lay the hemostats down away from the rat to pull the incision open. Repeat this process for each of the four corners, redoing any of the hemostats that have poor grip.
Drench the exposed skull with saline using cotton swabs. After soaking, wipe the area with dry cotton swabs and clean up any blood. Keeping the site as clean of blood as possible should allow the surgery to go more smoothly with less risk of infection.
Level the skull by adjusting the nose cone such that bregma and lambda are at equal height. There is a tool that can be placed onto the stereotax with three pins, two of which are calibrated to be the same height and separated by the length between bregma and lambda. Lower the pins to align with bregma and lambda on the skull, and align the skull such that the skull hits both points of the tool at the same time when it is lowered down. Using a magnifying glass can aid in this process. You can also use a drill bit for this process: lower the drill bit onto either bregma or lambda so that the bit is just barely touching the skull and note the position on the stereotax. Move the drill bit to the opposite landmark and do the same. Level the skull with the nosecone until the two landmarks measure to be within 50 microns of the same height. It is important to have the skull level so that we can most accurately hit the surgery coordinates which are based off bregma and lambda being leveled.
Measure out your surgery coordinates and mark the implant site by drilling a small, but visible hole. You can also use a marker to label the site.
Apply anchor screws. These are the pillars of the implant, so make sure each corner of the drive will be supported. You'll need at least 3-4 anchor screws around the exposed site to accomplish this. Don't use more screws than you need, but don't be afraid to use additional screws if any others aren't very secure. Have anchor screws as lateral as possible where the bone is thicker so there is more room to secure the screw. Also, leave plenty of room for the implant site and for the ground screw.
To drill the holes, use the HP3 drill bit. Make sure the drill is fastened securely into the stereotax so that it doesn't wobble when drilling the skull (this will blow out the hole and cause it to be too large to use). Drill the hole down until right before it fully penetrates the skull. It's fine if you do penetrate the skull, but it brings a new access point to the brain which increases risk of infection. The hole should be deep enough such that there are clear, straight edges along the side. The bottom of screw is flat while the bottom of the drill bit is beveled -- the screw can't bite into the beveled bottom, so straight edges are needed to keep the screw secure. Screw in each anchor screw by hand using the flat-head screwdriver. The drilled hole is smaller than the outer radius of the screws ridges, so it'll take a bit of force to twist down, but this is necessary for the screw to bite into the skull and hold securely in place. Note how many ridges equal the depth of the hole so that you don't drill it down too far. Use the tweezers to gently wiggle the screw after it has been inserted to make sure it's strongly fastened.
Acrylic the ground screws together, encasing each screw in acrylic. Make sure the acrylic is liquidy enough so it easily pours around the screws, but not so liquidy that it runs everywhere. Cover exposed skull, but leave room for the implant and the ground screw. Avoid getting acrylic on the skin and fur.
Shave down the bone at implant site (craniotomy). The technique for this depends on the type of implant you're doing. In general, you'll want to shave the bone down equally over the entire site until a thin layer of skull remains (it will begin to look transparent). At this point, drill through around the edges of the site such that you're left with a single piece of bone that can be pulled out with a tweezers.
Drill the hole for the ground screw. This will be done by hand like the craniotomy for the implant site; this time the hole needs to be larger than the screw so that the ground screw can slide in and rest atop the brain.
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After you are done with your specific procedure, suture the animals skin back together, then clean off any dried blood around the area with saline or ethanol.
Add antibiotic and pain reliving spray
To be continued.........................
After the crainiotomy, place a small piece of gel-foam over the exposed dura
Place a 10ul Hamilton syringe into the sterotax, be careful with the plastic components and plunger
Sterilize needle in bead sterilizer, then screw into the syringe
Get thawed virus in eppendorph tube, centrifuge if needed to get virus at the bottom
Place the tube in a holder away from the animal, and then position the needle above the tube in the center
Slowly lower the needle of the tube so it is as close to the tubes bottom without touching
Slowly draw up as much virus as needed, WITHOUT DRAWING AIR INTO THE NEEDLE
Withdraw a small amount of virus until a bubble is visible at the needle tip, then gently wipe this with the back of your glove
Next, go back to your crainiotomy and remove the foam, then nick the dura for ease of injection
Orientate the needle at the coordinates or the center of the hole and right above the brain.
SLOWLY lower the needle down to brain area of interest, on the order of microns per second
Once at area, lower the needle another 50um down, and then back up to original position prevent drift and pressure.
Now you can inject, (For mice GCaMP we do .5ul @ .05ul per minute)
Wait 5minutes after the injection is finished, then slowly pull up the needle
Once out of the brain, withdraw a small amount of virus to confirm virus injection
Clean needle and syringe with DH20, 4-5 times
Place all the virus tubes and weigh boats into a safety bag