Intracranial Pharmacotherapy and Pain Assays in Rodents

Summary

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Abstract

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Introduction

The central nervous system is known to play a key role in pain regulation. For example, glutamate signaling in the brain has a regulatory role in the context of pain^1,2. Hence, there is a need to study cellular and molecular signaling pathways in the brain with respect to pain. In addition, there is a need to understand if molecular targets in specific brain regions can be modified to treat pain. Current studies of pain in the brain rely on in vitro studies of electrophysiology in combination with systemic (intraperitoneal) delivery of pharmacological agents. In vitro studies have obvious deficits in revealing in vivo pain mechanisms. Meanwhile, systemic drug delivery does not delineate the precise cellular targets. In vivo intracranial injections of chemical and biologic agents have become a powerful tool to study neurological and molecular pathways in the brain. In recent years, other fields have used in vivo intracranial injections to successfully study addiction and reward behaviors and circuit pathways in rodents^3,4. However, in the context of pain, the use of in vivo intracranial pharmacology is lacking.

Intracranial injections allow for precise injection of a drug into a specific area of the brain. Furthermore, specific pathways and receptors can be targeted using highly selective drugs. The combination of an intracranial delivery system with precision drugs allows us to target molecular and cellular targets for pain. After intracranial delivery of these drugs, researchers can observe the immediate effects in the behavior of rodents. From well conducted experiments, rodents' behaviors can be linked with pharmacology.

In this protocol, we used the example of AMPAkine infusion in the prefrontal cortex (PFC) to demonstrate the mechanism of cortical glutamate signaling in pain regulation. AMPAkines are synthetic compounds that are known allosteric modulators. They have shown the ability to relieve acute and chronic pain in animal models^5,6. Previous studies suggest that the likely sites of action of AMPAkines are in the brain^5,6. The PFC is a region in the brain that exhibits top down control to subcortical areas to regulate mood and behavior. Some of these output projections have been shown to be key in pain regulation^1,2,7. More specifically, glutamate signaling in the PFC has been shown to regulate pain. Thus, the PFC was chosen as a targeted brain area for the study of AMPAkines in pain states.

Protocol

All procedures in this study were approved by the New York University School of Medicine Institutional Animal Care and Use Committee (IACUC) as consistent with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals.

1. Stereotaxic Cannula Implantation

1. Use 10-12 week old male Sprague-Dawley rats.
2. As previously described, anesthetize animals with 1.5-2% isoflurane^1,3,5. 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.
3. Stereotaxically implant bilateral 26-gauge guide cannulas into the PFC at a 12.5 degree angle with coordinates AP: +2.9 mm; ML: +/−1.6 mm; DV: −2.1 mm. In order to insert the cannulas, drill holes in the skull at the desired coordinates, with 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^1,2,7. To study the function of other specific brain areas, researchers can use different coordinates according to the brain atlas. For more details on performing Intracranial surgery please see Goffer et al (2013)³, Lee et al (2015)¹, and Sun et al (2017)¹⁰.
4. 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.
5. Once the animals have fully recovered from surgery, begin injections (next step).

2. Intracranial and Intraperitoneal Injections

1. For intracranial injections, use PE-50 tubing attached at one end to 10-μL Hamilton syringes with 33-gauge injector cannula that extend 1.0 mm beyond the implanted guides.
2. 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 species of animal.
   NOTE: Please note that saline should be used as a control instead of 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^8,9.
3. Inject volume bilaterally over a period of 100 s and keep injector cannulas in place for an additional 60 s prior to removal to allow slow diffusion of this solution.
4. 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)¹⁰.
   NOTE: It is recommended that intracranial injections are done first, as they are more difficult to perform than intraperitoneal injections.

3. Analgesia Assays and Assessment

1. To study the effect of intracranial injections on acute pain behavior in rats, use the plantar test (Hargreaves’ tests) 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.
   1. Begin by performing baseline Hargreaves’ tests prior to injections, to establish a baseline value for comparison.
2. Do not inject any drug of any kind prior to establishing a baseline value. Conduct 5 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.
3. Calculate withdrawal thresholds by taking the average of the 5 trials.
4. After obtaining a baseline average, begin the experiment to get withdrawal times after 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.
5. Calculate withdrawal thresholds as mentioned in step 3.2.

Results

As an example, we infused an AMPAkine into the PFC via cannulas (Figure 1). We also infused morphine systemically to assess the synergistic analgesic effects between AMPAkines and morphine. These results show that AMPAkines and morphine have an additive analgesic effect. It also shows that intracranial injections have the power to discover, at least in part, a mechanism for drug activation in the context of pain.

Figure 1. Intracranial injection of AMPAkines and systemic injection of morphine provide complementary analgesia. A graph comparing withdrawal latencies from Hargreaves' test after intracranial injections of either saline, AMPAkines, or AMPAkines with systemic injection of morphine. Error bars represent mean with SEM. n = 8; ****p < 0.0001, **p = 0.0099, *p = 0.0124, unpaired Student's t-test. This figure has been adapted from Sun et al. (2017)¹⁰, with permission from Elsevier. Please click here to view a larger version of this figure.

Discussion

In this study, we have demonstrated that intracranial pharmacology is a powerful tool to study pain mechanisms and has potential as a therapeutic delivery system. In our protocol, we delivered AMPAkines directly into the PFC and found that by enhancing glutamate signaling in the PFC, AMPAkines provided pain relief. We were able to demonstrate this through the use of intracranial injections combined with intraperitoneal injections, with subsequent pain assays. Based on the evidence of pain-relieving effects, when AMPAkines are delivered to the PFC, the current study suggests that the PFC may be involved as a target of AMPAkines. This is an important advantage of the intracranial pharmacological approach, when combined with behavior testing. In addition, the ability to combine intracranial with systemic delivery of drugs allows us to understand the therapeutic relationship between two different drugs and the potential pharmacologic interactions. In the example shown in this study, administering an AMPAkine into the PFC in combination with morphine shows an expected additive effect, which indicates that AMPAkines and morphine operate through different molecular mechanisms.

Although in vivo pharmacology is a powerful tool to study pain, it does have limitations. First, it is possible that, in inexperienced hands, the infused drug can diffuse to neighboring brain regions. This is a particular problem with mice. This could be overcome with the use of light-activated drugs and implantation of optic fibers, by measurement of drug levels at different distances from the injection site, or by injecting the drug at close anatomical sites. Second, intracranial injections can be short lasting, but the time period of effect isn't entirely known due to the pharmacokinetics of the drug. Other techniques, such as optogenetics, allow for instantaneous activation or inactivation of a desired region of the brain; this technique is more direct and has a known period of effect. On the other hand, in vivo pharmacology can target specific receptors or signaling pathways, and thus provide an additional level of molecular specificity. Thus, in the future, it will be necessary to explore combining in vivo pharmacology with additional techniques, such as in vivo physiology and optogenetics. With the combination of these tools, new pathways and receptor specific pathways can be discovered and used to treat pain.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sterotaxic Cannula	PlasticsOne	8I26GA8MMKIT
Digital Syringe	Hamilton	8440
AMPAkine	Sigma Aldrich	C-271
Dimethyl Sulfoxide	Sigma Aldrich	D4540
Hargreaves Apparatus	Ugo Basile	37370
Male Sprague-Dawley rats	Taconic Farms	NTac:SD
Sterile Surgical gloves	Dynarex	6535