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| intra-peritoneal (into central body cavity through abdomen) |
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| intramuscular (muscle of arm/leg) |
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| Put on lab coats, familiarize with lab materials and apparatuses. Wear gloves when handling animals |
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| 250 gram wistar rats, in covered cages, don't play/excessively handle (affects anesthetic). Instructor will inject rats with acepromazine (2mg/kg) 20mins before student handling: sedative/preanesthetic reduces stress, helps anesthesia and recovery from it |
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| find and identify surgical instrument pack (don't open until told) |
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| find and identify materials like eye ointment and betadine (iodine soln) to be used before/during surgery |
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| w/Dale's help, identify antibiotic enrofloxacin (Baytril) and anesthetic pentobarbital. calculate appropriate volume |
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| Anesthetize and prepare the animal |
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| with a loaded syringe of anesthetic available, remove animal from cage and make in IP injection as demonstrated by instructor. Handle rat firm but gentle, use tail to orient, grasp behind head and around anterior. control head to prevent biting. assistant holds hind legs away from abdomen. make quick, smooth injection, then replace animal in cage. record time and dose of administration of anesthetic. be ready to boost animal later with 1/3 of original dose IM as required (after about an hour) |
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| as soon as unresponsive, remove from cage. check anesthesia by pinching the webbing of hind foot and watch for withdrawal (should not). continue to monitor this way for boosts. only boost for movements that are in response to your stimulus. expect a few minute delay between boost and return to deep anesthesia. work quickly, check every 10 min or if unusual activity is noted. make records of times doses, assessments, and actions |
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Definition
| apply eye ointment to the eyes to prevent drying of cornea |
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| inject animal with antibiotic IM (large muscles of hind limb) |
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Definition
| closely shave entire top of head with clippers from between eyes to back of the head |
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Definition
| label tail with appropriate markings |
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| place animal in stereotaxic apparatus |
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| Place an insulating pad on the base plate of the stereotaxic apparatus. Place a preheated, phase-transition warming pad on the insulating layer and place the animal on the warming pad. As soon as the animal is deeply anesthetized, the body temperature will begin to drop unless external heat is supplied. If this is allowed to continue, the likelihood of losing the animal is significant. The warming pad should be good for the entire surgery. |
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Definition
| Fix and center the head of the animal in the apparatus with the ear bars. An instructor will help with this tricky procedure. When completed, this process should result in the animal’s head being fixed so that it rotates only in the sagittal plane. |
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Definition
| Gently pry open the mouth of the animal, keeping your fingers away from the teeth, and place the incisor bar behind the animal’s upper incisors. apply very gentle tension to the incisors and fix the bar in place in the anterior-posterior aspect. gently tighten the nose clamp to hold the nose in place over the incisor bar. Adjust the height of the nose clamp/incisor bar to level top of the head of the animal so that the imagined line between lambda and bregma appears parallel to the horizontal parts of the apparatus. (You will make fine adjustments later.) |
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| Inject 1 ml of sterile saline subcutaneously (S.C.) in the neck region of the animal and continue these injections at 1 hour intervals throughout the procedure to prevent dehydration of the animal. |
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| Expose the skull of the animal |
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| Scrub the shaved area of the head with betadine and dry well with a gauze pad. (The adhesive surgical drape will not adhere to a wet surface) |
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Definition
| At this time, the designated surgical member of the group should put on a mask, bonnet, and sterile gloves and avoid touching non-sterile areas. A second designated member should open the sterilized foil pack of instruments and help create a sterile field for the surgeon. The third member of the group will be keeping records and testing the depth of anesthesia at 10-15 minute intervals. |
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Term
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Definition
| Place an adhesive sterile drape over the animal with assistance from the instructors. Only a “sterile” member of the group is allowed to touch the top of the drape where it covers the animal. Gently but firmly adhere the drape to the top of the animal’s head. Leave access to the rear paws of the animal for testing of levels of anesthesia. The easiest way to do this is to leave the backing on the drape in this area. |
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Definition
| Infiltrate about 50 l of 2% lidocane (local anesthetic) under the skin that covers the top of the skull. Do this by pushing a fine syringe needle under the skin for some distance and then injecting while slowly withdrawing the needle with side to side movement of the tip. |
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| Using a scalpel, make a firm, sagittal-plane incision in the skin over most of the length of the shaved skull. Use sufficient force with the blade against the skull to cut completely through the skin. |
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| Reflect the sides of the cut with hemostats and remove connective tissue from the skull with a dull scraper blade. Finish by scrubbing the surface with sterile swabs. As the skull surface dries slightly, the suture lines will become clearly visible. |
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| set up the stereotaxic apparatus |
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| Mount the cannula guide in the arm of the manipulator if it is not already there (check stability). |
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Definition
| Mount the manipulator arm on the stereotax and fix in place. Be sure that the zero mark on the arm is aligned with the reference mark on the apparatus. |
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Definition
| Move the tip of the cannula guide back and forth between lambda and bregma and adjust the incisor bar until the two landmarks are at the same height. make sure that the tip of the guide just touches but does not depress the surface of the skull when these measurements are being made. |
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| Move the cannula guide tip to the starting landmark and determine the three manipulator readings. |
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Definition
| Calculate the proper manipulator readings in the anterior-posterior and medial-lateral directions in order to place the tip directly over the first target of interest, move the tip to this position and mark the skull directly under the tip. |
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Definition
| Repeat steps b. Through d. for the second target. |
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Definition
| Ask an instructor to assist you in boring holes in the skull at your marked points. You do not need to remove the manipulator arm to drill holes. Just raise the guide well away from the skull. |
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| Obtain a syringe/cannula filled with dye from the instructor in charge of dyes. |
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| Push the cannula into the cannula guide until it is captured by the guide. Support the guide assembly so your pressure does not cause it to slip in the clamp – only gentle pressure is required The tip of the cannula will extend 1mm out from the tip of the guide. |
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| Position the cannula tip over the appropriate starting landmark. Be careful not to dislodge the cannula from the guide. Note the manipulator positions. Your dorsal/ventral reading should be about 1mm higher than before. Move the tip to the appropriate spot over the skull as you did when marking the skull. This should now be over the drilled hole. |
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Definition
| Determine the dorsal-ventral manipulator reading for your final target position and move to that point. It may be helpful to overshoot the target very slightly and withdraw to the correct position, particularly in the case of cortical targets. |
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Definition
| Inject 0.1 to 2.0 l of dye as instructed. Inject slowly! (Inject 0.1 -0.2 l, wait 30 seconds, repeat until proper amount of dye is injected.) |
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| Withdraw the cannula guide from the brain gently and slowly with the manipulator |
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| Gently remove the cannula from the cannula guide with forceps. |
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Definition
| Rinse the surface of the brain with sterile saline. |
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| Return the used syringe/cannula to the instructor in charge of dyes and rinse out the cannula guide as instructed to avoid dye cross contamination of injection sites. |
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| close surgical wound and allow animal to recover |
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| Fill holes in skull with gelfoam. |
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| Apply lidocaine ( 200ul, 2%) solution to the cut regions of the scalp |
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| Suture the skin back over the skull. Begin in the center of the incision, taking care to bring the edged of the skin close together. Place additional sutures sequentially in the center of remaining gaps in the skin until the wound is fully closed. Work quickly and try not to boost the anesthetic at this point in order to enhance the animal’s chances of survival |
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Definition
| Remove the animal from the stereotaxic instrument and place in a labeled, covered recovery cage. |
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| With the help of the instructors, add Ibuprophen (0.4 mg/ml) to the animal’s drinking water to minimize pain associated with the animal’s wound. |
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| (Lab staff will monitor the animals until they have recovered from anesthesia.) |
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| Discard all contaminated materials and sharp disposables as instructed by lab staff. |
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| Wash instruments thoroughly to remove all blood and leave to air dry. |
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