Name: intracranial pharmacotherapy and pain assays in rodents
Uploaded: 2019-04-09T12:30:00.000+00:00
Duration: 2 min 26 s
Description: Watch this Scientific Journal Video about Intracranial Pharmacotherapy and Pain Assays in Rodents at JoVE.com

This work was supported by the National Institute of General Medical Sciences (GM102691, GM115384), National Institute of Neurological Disorders and Stroke (NS100065), (Bethesda, MD, USA) and the Anesthesia Research Fund of New York University Department of Anesthesiology (New York, NY, USA).

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
<ol>
	<li>Use 10-12 week old male Sprague-Dawley rats.</li>
	<li>As previously described, anesthetize animals with 1.5-2% isoflurane1,3,5. 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&#8217;s eyes to prevent damage.</li>
	<li>Stereotaxically implant bilateral 26-gauge guide cannulas into the PFC at a 12.5 degree angle with coordinates AP: +2.9 mm; ML: +/&#8722;1.6 mm; DV: &#8722;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 studies1,2,7. 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.</li>
	<li>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.</li>
	<li>Once the animals have fully recovered from surgery, begin injections (next step).</li>
</ol>
2. Intracranial and Intraperitoneal Injections
<ol>
	<li>For intracranial injections, use PE-50 tubing attached at one end to 10-&#956;L Hamilton syringes with 33-gauge injector cannula that extend 1.0 mm beyond the implanted guides.</li>
	<li>Inject 0.5 &#956;L (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 &#956;L, as in this case) may not interfere with behavior studies8,9.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Analgesia Assays and Assessment
<ol>
	<li>To study the effect of intracranial injections on acute pain behavior in rats, use the plantar test (Hargreaves&#8217; tests) to calculate the withdrawal latency in response to thermal stimuli. The Hargreaves&#8217; apparatus focuses an infrared beam through a glass plane onto the rat&#8217;s foot; the rat is standing and freely moving above the glass plane. When performing Hargreaves&#8217; test, focus the infrared beam under the plantar area of the rat&#8217;s foot.
	<ol>
		<li>Begin by performing baseline Hargreaves&#8217; tests prior to injections, to establish a baseline value for comparison.</li>
	</ol>
	</li>
	<li>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&#8217; 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.</li>
	<li>Calculate withdrawal thresholds by taking the average of the 5 trials.</li>
	<li>After obtaining a baseline average, begin the experiment to get withdrawal times after infusion of drugs. Hargreaves&#8217; 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.</li>
	<li>Calculate withdrawal thresholds as mentioned in step 3.2.</li>
</ol>

<ol>
	<li>Lee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+corticostriatal+circuitry+relieves+chronic+neuropathic+pain.">Activation of corticostriatal circuitry relieves chronic neuropathic pain.</a> The Journal of Neuroscience. 35 (13), 5247-5259 (2015).</li><li>Martinez, E., Lin, H. H., Zhou, H., Dale, J., Liu, K., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticostriatal+Regulation+of+Acute+Pain.">Corticostriatal Regulation of Acute Pain.</a> Frontiers in Cellular Neuroscience. 11, 146 (2017).</li><li>Goffer, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-permeable+AMPA+receptors+in+the+nucleus+accumbens+regulate+depression-like+behaviors+in+the+chronic+neuropathic+pain+state.">Calcium-permeable AMPA receptors in the nucleus accumbens regulate depression-like behaviors in the chronic neuropathic pain state.</a> The Journal of Neuroscience. 33 (48), 19034-19044 (2013).</li><li>Carr, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+subunit+GluR1+downstream+of+D-1+dopamine+receptor+stimulation+in+nucleus+accumbens+shell+mediates+increased+drug+reward+magnitude+in+food-restricted+rats.">AMPA receptor subunit GluR1 downstream of D-1 dopamine receptor stimulation in nucleus accumbens shell mediates increased drug reward magnitude in food-restricted rats.</a> Neuroscience. 165, 1074-1086 (2010).</li><li>Su, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPAkines+target+the+nucleus+accumbens+to+relieve+postoperative+pain.">AMPAkines target the nucleus accumbens to relieve postoperative pain.</a> Anesthesiology. 125 (5), 1030-1043 (2016).</li><li>Le, A. M., Lee, M., Su, C., Zou, A., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPAkines+have+novel+analgesic+properties+in+rat+models+of+persistent+neuropathic+and+inflammatory+pain.">AMPAkines have novel analgesic properties in rat models of persistent neuropathic and inflammatory pain.</a> Anesthesiology. 121 (5), 1080-1090 (2014).</li><li>Cooper, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetisation+of+prefrontal+cortex+and+response+to+noxious+stimulation.">Anaesthetisation of prefrontal cortex and response to noxious stimulation.</a> Nature. 254 (5499), 439-440 (1975).</li><li>Blevins, J. E., Stanley, B. G., Reidelberger, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMSO+as+a+vehicle+for+central+injections:+tests+with+feeding+elicited+by+norepinephrine+injected+into+the+paraventricular+nucleus.">DMSO as a vehicle for central injections: tests with feeding elicited by norepinephrine injected into the paraventricular nucleus.</a> Pharmacology Biochemistry and Behavior. 71, 277-282 (2002).</li><li>Schmeichel, B. E., Herman, M. A., Roberto, M., Koob, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocretin+Neurotransmission+Within+the+Central+Amygdala+Mediates+Escalated+Cocaine+Self-administration+and+Stress-Induced+Reinstatement+in+Rats.">Hypocretin Neurotransmission Within the Central Amygdala Mediates Escalated Cocaine Self-administration and Stress-Induced Reinstatement in Rats.</a> Biological Psychiatry. 81, 606-615 (2017).</li><li>Sun, Y., Liu, K., Martinez, E., Dale, J., Huang, D., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPAkines+and+morphine+provide+complementary+analgesia.">AMPAkines and morphine provide complementary analgesia.</a> Behavioural Brain Research. 334 (2017), 1-5 (2017).</li></ol>

The authors have nothing to disclose.

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&#39;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.

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 pain1,2. 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 rodents3,4. 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&#39; 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 models5,6. Previous studies suggest that the likely sites of action of AMPAkines are in the brain5,6. 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 regulation1,2,7. 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.

<table><tbody><tr><td>Sterotaxic Cannula</td><td>PlasticsOne</td><td>8I26GA8MMKIT</td><td></td></tr><tr><td>Digital Syringe</td><td>Hamilton</td><td>8440</td><td></td></tr><tr><td>AMPAkine</td><td>Sigma Aldrich</td><td>C-271</td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma Aldrich</td><td>D4540</td><td></td></tr><tr><td>Hargreaves Apparatus</td><td>Ugo Basile</td><td>37370</td><td></td></tr><tr><td>Male Sprague-Dawley rats</td><td>Taconic Farms</td><td>NTac:SD</td><td></td></tr><tr><td>Sterile Surgical gloves</td><td>Dynarex</td><td>6535</td><td></td></tr></tbody></table>

intracranial pharmacotherapy and pain assays in rodents

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.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58473/58473fig1.jpg" /> 
Figure 1. Intracranial injection of AMPAkines and systemic injection of morphine provide complementary analgesia. A graph comparing withdrawal latencies from Hargreaves&#39; test after intracranial injections of either saline, AMPAkines, or AMPAkines with systemic injection of morphine. Error bars represent mean with SEM. n = 8; ****p &#60; 0.0001, **p = 0.0099, *p = 0.0124, unpaired Student&#39;s t-test. This figure has been adapted from Sun et al. (2017)10, with permission from Elsevier. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58473/58473fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Intracranial Pharmacotherapy and Pain Assays in Rodents at JoVE.com

Intracranial Pharmacotherapy and Pain Assays in Rodents

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.

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

intracranial-pharmacotherapy-and-pain-assays-in-rodents

Nanyang Technological University

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Unit 1

Drugs for Pain Management: Opioid Analgesics and General Anesthetics

SCIENTISTS IN ACTION

5.2K Views.  New York University School of Medicine. Het belangrijkste voordeel van deze techniek is dat het kan worden gebruikt om verder te verkennen pijnmechanismen en drugsacties in de hersenen. Om het effect van intracraniale injecties op acuut pijngedrag bij ratten te bestuderen, een week na de operatie, plaats de rat op de glasplaat van een Hargreaves-apparaat en gebruik het apparaat om een infraroodstraal door het glazen vlak onder het plantaire gebied van een van de voeten van de rat te concentreren. Om de synergetische farmacologische...

Video: Intracranial Pharmacotherapy and Pain Assays in Rodents

Determining Pain Detection and Tolerance Thresholds Using an Integrated, Multi-Modal Pain Task Battery

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify potentially suitable therapeutic populations. The need to use a comprehensive battery of pain models is highlighted by studies whereby only a single pain model, thought to relate to the clinical situation, demonstrates lack of efficacy. No single experimental model can mimic the complex nature of clinical pain. The integrated, multi-modal pain task&#160;battery presented here encompasses the electrical stimulation task, pressure stimulation task, cold pressor task, the UVB inflammatory model which includes a thermal task and a paradigm for inhibitory conditioned pain modulation. These human pain models have been tested for predicative validity and reliability both in their own right and in combination, and can be used repeatedly, quickly, in short succession, with minimum burden for the subject and with a modest quantity of equipment. This allows a drug to be fully characterized and profiled for analgesic effect which is especially useful for drugs with a novel or untested mechanism of action.

Human pain models are valuable tools used to assess the analgesic potential of novel compounds and predict their clinical efficacy, especially when used in an integrated manner. Although implementation of these models is complex, with proper execution, the pain models described in this protocol can provide predictive and reliable results.

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify ...

Behavior

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. 
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of na&#239;ve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as ...

Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results by addressing the cognitive and motivational aspects of pain in rodents. This paper presents a protocol for assessing mechanical hypersensitivity following chronic constriction injury of the infraorbital nerves (CCI-ION) in rats using an orofacial operant pain system. Before CCI-ION surgery, rats were trained in an orofacial pain assessment device (OPAD) to drink sweetened condensed milk while making facial contact with the metal spiked bars and lick-tube.
In this assay, rats can choose between receiving milk as a positive reinforcer or escaping an aversive mechanical stimulus that is produced by a vertical row of small pyramid-shaped spikes on each side of the reward access hole. Following 2 weeks of training in the OPAD and before the CCI-ION surgery, baseline mechanical sensitivity data were recorded for 5 days for each rat during a 10 min testing session. During a session, the operant system automatically records the number of reward bottle activations (licks) and facial contacts, contact duration, and latency to the first lick, among other measures.
Following baseline measurements, rats underwent either CCI-ION or sham surgery. In this protocol, mechanical hypersensitivity was quantified by measuring the number of licks, latency to the first lick, the number of contacts, and the ratio of licks to facial contacts (L/F). The data showed that CCI-ION resulted in a significant decrease in the number of licks and the L/F ratio and an increase in the latency to the first lick, indicating mechanical hypersensitivity. These data support the use of operant-based pain assays to assess mechanical pain sensitivity in preclinical pain research.

This protocol describes the assessment of mechanical hypersensitivity in a rat model of neuropathic orofacial pain using an operant-based orofacial pain assessment device.

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results ...

Real-Time Mouse Eye Squint Detection for Migraine Pain Tracking Using DeepLabCut

An Automated Squint Method for Time-syncing Behavior and Brain Dynamics in Mouse Pain Studies

Spontaneous pain has been challenging to track in real time and quantify in a way that prevents human bias. This is especially true for metrics of head pain, as in disorders such as migraine. Eye squint has emerged as a continuous variable metric that can be measured over time and is effective for predicting pain states in such assays. This paper provides a protocol for the use of DeepLabCut (DLC) to automate and quantify eye squint (Euclidean distance between eyelids) in restrained mice with freely rotating head motions. This protocol enables unbiased quantification of eye squint to be paired with and compared directly against mechanistic measures such as neurophysiology. We provide an assessment of AI training parameters necessary for achieving success as defined by discriminating squint and non-squint periods. We demonstrate an ability to reliably track and differentiate squint in a CGRP-induced migraine-like phenotype at a sub second resolution.

Author Spotlight: Deciphering Electrical Networks Behind Complex Brain Activities and Disorders

This protocol provides a method for tracking automated eye squint in rodents over time in a manner compatible with time-locking to neurophysiological measures. This protocol is expected to be useful to researchers studying mechanisms of pain disorders such as migraine.

Spontaneous pain has been challenging to track in real time and quantify in a way that prevents human bias. This is especially true for metrics of head ...

Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral procedures to study trigeminal neuropathic pain in rats. To meet the specificity of trigeminal neuropathic pain syndromes, the infraorbital nerve (IoN) is subjected to a chronic constriction injury (CCI) by loosely ligating the nerve. An intra-orbital approach is presented here to expose and ligate the IoN in the orbital cavity. After IoN ligation, rats exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are indicative of persistent pain and mechanical allodynia. Two phases can be defined in the development of the behavioral changes. During the first week following IoN-CCI (phase 1), rats show an increased and asymmetric face grooming activity, i.e., with face wash strokes primarily directed to the nerve-injured IoN territory. A distinction is made between face grooming behavior that is part of a more general body grooming behavior, which remains largely unaffected by IoN-CCI, and face grooming that is neither preceded nor followed by body grooming, which is significantly increased after IoN-CCI. During this period, responsiveness to mechanical stimulation of the IoN territory is reduced. This hyporesponsiveness is abruptly replaced by an extreme hyperresponsiveness whereby even very weak stimulus intensities provoke nocifensive behavior (phase 2). The phenomenological similarities between these behavioral alterations and reported signs of facial pain (i.e., responses to noxious stimulation of the face) suggest the presence of dysesthesia/paresthesia and mechanical allodynia in the ligated IoN territory.

Chronic Constriction Injury of the Rat's Infraorbital Nerve IoN-CCI to Study Trigeminal Neuropathic Pain

This manuscript describes a rodent model to study trigeminal neuropathic pain. The methods described include surgical procedures to perform a chronic constriction injury of the rat&#8217;s infraorbital nerve and the post-surgical behavioral tests to measure the changes in spontaneous and evoked behavior that are indicative of persistent pain and allodynia.

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral ...

Medicine

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

Dural Stimulation and Periorbital von Frey Testing in Mice As a Preclinical Model of Headache

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system. For example, they protect the brain from the skull and anchor/organize the vascular and neuronal supply of the cortex. However, the meninges are also implicated in nervous system disorders such as migraine, where the pain experienced during a migraine is attributed to local sterile inflammation and subsequent activation of local nociceptive afferents. Of the layers in the meninges, the dura mater is of particular interest in the pathophysiology of migraines. It is highly vascularized, harbors local nociceptive neurons, and is home to a diverse array of resident cells such as immune cells. Subtle changes in the local meningeal microenvironment may lead to activation and sensitization of dural perivascular nociceptors, thus leading to migraine pain. Studies have sought to address how dural afferents become activated/sensitized by using either in vivo electrophysiology, imaging techniques, or behavioral models, but these commonly require very invasive surgeries. This protocol presents a method for comparatively non-invasive application of compounds on the dura mater in mice and a suitable method for measuring headache-like tactile sensitivity using periorbital von Frey testing following dural stimulation. This method maintains the integrity of the dura and skull and reduces confounding effects from invasive techniques by injecting substances through a 0.65 mm modified cannula at the junction of unfused sagittal and lambdoid sutures. This preclinical model will allow researchers to investigate a wide range of dural stimuli and their role in the pathological progression of migraine, such as nociceptor activation, immune cell activation, vascular changes, and pain behaviors, all while maintaining injury-free conditions to the skull and meninges.

The most notable symptom of migraine is severe head pain, and it is hypothesized that this is mediated by sensory neurons innervating the meninges. Here, we present a method to locally apply substances to the dura in a minimally invasive manner while using facial hypersensitivity as an output.

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system.  ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

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