All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Stereotaxic Cannula Implantation
- Use 10-12 week old male Sprague-Dawley rats.
- Anesthetize animals with 1.5-2% isoflurane. Once the animal becomes unresponsive to a strong pinch with sharp forceps, perform the surgery. Be sure to autoclave all instruments, use sterile surgical gloves, and add ophthalmic ointment to the animal’s eyes to prevent damage.
- Stereotaxically implant bilateral 26-gauge guide cannulas into the prefrontal cortex (PFC) at a 12.5 degree angle with coordinates anteroposterior (AP): +2.9 mm; mediolateral (ML): +/−1.6 mm; dorsoventral (DV): −2.1 mm. In order to insert the cannulas, drill holes in the skull at the desired coordinates, with a hole diameter according to the size of the cannulas used.
NOTE: Here, the PFC was studied as the potential target for intracranial implants because of its important role in pain processing, which has been demonstrated in a number of previous studies. To study the function of other specific brain areas, researchers can use different coordinates according to the brain atlas. - Allow rats to recover from surgery for at least 1 week. Following surgery, inject subcutaneous fluids prior to recovery to help support the metabolic requirements, and apply topical bupivacaine in the freshly closed incision. Place the animal on a warm pad until they wake up, and monitor the animal post operation for 3 days to ensure good health and a proper recovery.
- Once the animals have fully recovered from surgery, begin the injections (next step).
2. Intracranial and Intraperitoneal Injections
- For intracranial injections, use PE-50 tubing attached at one end to 10-μL Hamilton syringes with a 33-gauge injector cannula that extends 1.0 mm beyond the implanted guides.
- Inject 0.5 μL (or less if desired) of either the study drug or saline in the PFC of these rats. Because the PFC is a larger region in rats, this amount will not be spread to other regions. However, for smaller brain regions or for mice, use a smaller volume. The amount injected will depend on the region of the brain and the species of animal.
NOTE: Please note that saline should be used as a control instead of dimethyl sulfoxide (DMSO) because DMSO is neurotoxic. However, a very small amount of DMSO may be safe for infusion, as studies have shown that less than 50% DMSO (i.e., less than a total volume of 0.3 μL, as in this case) may not interfere with behavior studies. - Inject volume bilaterally over a period of 100 s and keep the injector cannulas in place for an additional 60 s prior to removal to allow slow diffusion of this solution.
- For studies of synergistic pharmacological effects, co-administer another drug through systemic methods. In this case, inject the desired drug or control intracranially and administer an additional drug intraperitoneally immediately thereafter. As an example, in this study, we studied the synergistic analgesic effects of morphine and Ampakines to test for an additive effect. We infused an AMPAkine intracranially, in combination with intraperitoneal delivery of 1 mg/kg morphine (a safe systemic dose).
NOTE: It is recommended that intracranial injections be done first, as they are more difficult to perform than intraperitoneal injections.
3. Analgesia Assays and Assessment
- To study the effect of intracranial injections on acute pain behavior in rats, use the plantar test (Hargreaves’ test) to calculate the withdrawal latency in response to thermal stimuli. The Hargreaves’ apparatus focuses an infrared beam through a glass plane onto the rat’s foot; the rat is standing and freely moving above the glass plane. When performing Hargreaves’ test, focus the infrared beam under the plantar area of the rat’s foot.
- Begin by performing baseline Hargreaves’ tests prior to injections to establish a baseline value for comparison.
- Do not inject any drug of any kind prior to establishing a baseline value. Conduct five good trials, 5 min apart. A good trial is indicated by a clear withdrawal, i.e., when the rat bends its knee and lifts its foot upward and into the body.
NOTE: Make sure the trials are 5 min apart to prevent sensitization of the rat. The Hargreaves’ apparatus automatically records the time of withdrawal, once the infrared beam is broken. As a result, be sure to discount trials from locomotion, shifting weight, etc. If a discounted trial occurs, wait 5 min and repeat the trial. - Calculate withdrawal thresholds by taking the average of the five trials.
- After obtaining a baseline average, begin the experiment to get withdrawal times after the infusion of drugs. Hargreaves’ tests can be performed 20-30 min after intracranial injections, although the precise timing may be dependent on the pharmacokinetics of the specific agents. This is to ensure that the rat has absorbed the drug and is experiencing its effects. Conduct this experiment in the same manner as step 3.1.
- Calculate withdrawal thresholds as mentioned in step 3.2.
