Neurobiological studies of chronic pain and analgesia: Rationale and refinements.

European Journal of Pharmacology 759 (2015) 169–181

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Neurobiological studies of chronic pain and analgesia: Rationale and refinements Carolyn A. Fairbanks a,b,c,n, Cory J. Goracke-Postle d a

University of Minnesota, Department of Pharmaceutics, Minneapolis, MN, USA University of Minnesota, Department of Pharmacology, Minneapolis, MN, USA c University of Minnesota, Department of Neuroscience, Minneapolis, MN, USA d University of Minnesota, Office of the Vice President for Research, Minneapolis, MN, USA b

art ic l e i nf o

a b s t r a c t

Article history: Received 29 January 2015 Received in revised form 5 March 2015 Accepted 12 March 2015 Available online 24 March 2015

Chronic pain is a complex condition for which the need for specialized research and therapies has been recognized internationally. This review summarizes the context for the international call for expansion of pain research to improve our understanding of the mechanisms underlying pain in order to achieve improvements in pain management. The methods for conducting sensory assessment in animal models are discussed and the development of animal models of chronic pain is specifically reviewed, with an emphasis on ongoing refinements to more closely mimic a variety of human pain conditions. Pharmacological correspondences between pre-clinical pain models and the human clinical experience are noted. A discussion of the 3Rs Framework (Replacement, Reduction, Refinement) and how each may be considered in pain research is featured. Finally, suggestions are provided for engaging principal investigators, IACUC reviewers, and institutions in the development of strong partnerships to simultaneously expand our knowledge of the mechanisms underlying pain and analgesia while ensuring the humane use of animals in research. & 2015 Elsevier B.V. All rights reserved.

Keywords: Chronic pain Analgesia Animal models Reduction Replacement Refinement

1. Introduction 1.1. Chronic pain is a global health concern Chronic pain is recognized as a global problem and control of pain through both non-pharmacologic and pharmacologic approaches is a shared international goal. Since its establishment in 1974, the International Association for the Study of Pain (IASP) has provided a consistent and ongoing international forum, for health care professionals and scientists representing 133 countries, to work toward advancing the scientific understanding of pain, guiding improvements in pain management practices, and providing education on pain and analgesia. In 1986 the World Health Organization (WHO) introduced the Analgesic Ladder, which provided a unifying framework for the treatment of chronic pain. The WHO Analgesic Ladder was translated into 22 languages and served as an essential tool in the advocacy for adequate pain control (Vargas-Schaffer, 2010). Since its introduction, it has contributed an essential foundation upon which modifications n Correspondence to: University of Minnesota, College of Pharmacy, 9-177, Weaver Densford Hall, 308 Harvard Street S.E. Minneapolis, MN 55455, USA. Tel. þ1 612 625 2945. E-mail address: [email protected] (C.A. Fairbanks).

http://dx.doi.org/10.1016/j.ejphar.2015.03.049 0014-2999/& 2015 Elsevier B.V. All rights reserved.

representing new treatment approaches and/or adaptations in diagnosis continue to be considered and integrated. In October of 2000, the 106th United States Congress passed H.R. 3244 and, through President Clinton subsequently signing this bill into law, 2001–2010 was declared the Decade of Pain Control and Research (Title VI, section 1603). As was the case with the previous decade, the Decade of the Brain (1990–2000), the expectation was that greater awareness of the problem of pain would be generated and that additional funding for pain research would be prioritized (Nelson, 2003). In January of 2001, the Joint Commission on Accreditation of Health Care Organizations (JCAHO) established a new mandate that pain be considered a vital sign (Lanser and Gesell, 2001); this concept was initiated in the mid-1990s by the leadership of the American Pain Society (Campbell, 1996) through an extensive educational campaign to require health care professionals to assess pain level in all patients in the same manner as temperature, blood pressure, pulse, and respiratory rate (Lanser and Gesell, 2001) and provide treatment. Expanding pain assessments has since greatly raised awareness of the prevalence of pain, while simultaneously uncovering the challenge of how to effectively and safely manage pain (Morone and Weiner, 2013). Building on the momentum of increased recognition of the problem of pain, the WHO, the IASP, and the European Federation of IASP Chapters (EFIC) held the first Global Day Against Pain in

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C.A. Fairbanks, C.J. Goracke-Postle / European Journal of Pharmacology 759 (2015) 169–181

Geneva Switzerland in 2004 (Lipman, 2005). The call for recognition of pain control as a human right was introduced there by the IASP leadership. This forum continued the momentum of increasing recognition of the problem of chronic pain. On October 11, 2010, the IASP, WHO, and EFIC, held a second Global Day Against Pain (International Pain Summit of the International Association for the Study of Pain, 2011). This forum was attended by 230 representatives from IASP chapters in 64 distinct countries and included IASP members from 130 countries (Cousins and Lynch, 2011). Through this assembly, the international membership of the IASP codified its commitment to global pain management through the “Declaration of Montreal” which asserts that access to pain management is a fundamental human right (Cousins and Lynch, 2011). Shortly thereafter, the Institute of Medicine (IoM) published a now widely cited report (IOM, 2011) on the state of chronic pain in the United States (U.S.). Authored by a panel of well recognized pain management and research experts, the report features the current experience of pain, education needs for specific populations with respect to pain, and challenges for research on pain and development of novel analgesics. This report is the source of the often-cited figures that 100 million U.S. adults are afflicted with chronic pain and that there is an estimated cost of $560–635 billion/year associated with chronic pain. This document, therefore, provides a unifying blueprint for the clinical and basic pain research community to raise awareness regarding pain management and advocate for strategic support for pain research. Through featuring the timeline of global recognition of the problem of pain, the need for pain management, and the need for in-depth mechanistic research on pain mechanisms and analgesic targets, the rationale for the importance of pain research is established. 1.2. Defining chronic pain The authors of the IoM Blueprint on Chronic Pain recognized that significant advances have already been made in understanding the basic mechanisms underlying pain. Acknowledgment of the progress made establishes a solid basis for the proposal to expand our understanding of the complexity of chronic pain states. A very large portion of the knowledge currently established was achieved through the use of animals in research, primarily (but not exclusively) rodents. Just as the term cancer applies to a broad spectrum of conditions associated with pathological cellular proliferation, the term chronic pain describes diverse conditions associated with maladaptive sensory input of varying etiologies. Unlike many disease pathologies, the condition of pain frequently accompanies a disease as a symptom and is often the precipitating event that motivates an individual to seek medical evaluation. However, established unrelieved chronic pain is often recognized as a disorder in and of itself, independent of a primary disorder. Much education and advocacy during the last several decades has resulted in recognition of chronic pain as a spectrum of sensory disorders requiring treatment. At the foundation of that advocacy in many ways resides an essential body of knowledge that has arisen through strategic and diligent neurophysiological, neuroanatomical, and neuropharmacological studies to define the sensory coding of the pain pathways, to map the expression of pain and analgesia relevant systems throughout the central and peripheral nervous systems, and to identify the responses of living organisms to analgesic agents that inhibit the pain signal at various points throughout the pain pathway. The vast majority of what the pain research community has uncovered regarding the pain pathways and analgesic systems is attributed in very large part to the use of animal subjects through application of a wide variety of focused stimuli applied to activate sensory nerve endings, astute observation of spontaneous behaviors, and development of chronic pain models to optimize simulation of the human clinical pain experience to the greatest extent possible. We are indebted to the committed diligence of

several academic generations of an international pain research community who established this foundation. Through that collective effort, a substantive understanding of how pain is transmitted through the mammalian system and how the endogenous inhibitory systems provide opportunities for analgesia has been achieved. This review will feature how pain is assessed, including the specific approaches used to study various forms of chronic pain and the refinements that have been implemented. Additionally, there will be discussion of the translation of this knowledge, how it corresponds to human pain systems, and how it has advanced therapeutic treatments, including intervention strategies at distinct relays in the pain pathway. Further, there will be review of the new approaches that are increasingly incorporated into scientific designs with the intention of optimizing and more closely modeling the human clinical pain condition. The final section will address how the 3Rs (Replacement, Reduction, Refinement) may be considered in the context of pain research.

2. Primary afferent sensory neurons and the pain signal transmission pathway The vast majority of studies of pain in animal subjects have used a variety of focused stimuli that are intended to activate sensory nerve endings. These reflex measures have been beautifully summarized recently in the Journal of Pain and the reader is referred to Gregory and colleagues (Gregory et al., 2013) for a more in-depth explanation. However, to appreciate the utility of the activation of these sensory neurons, it is important to understand how they carry the pain signal forward. Pain sensation is encoded primarily by sensory neurons with axons classified as thinly myelinated A delta and unmyelinated C fibers. The cell bodies of these neurons are located within the dorsal root ganglia and emanate a single process that bifurcates into a peripheral and central component. The terminals of the peripheral process innervate peripheral targets such as skin or visceral tissues. These terminals host a variety of ion channels and receptors in their bilipid membrane that are designed to detect a wide variety of potentially damaging stimuli, such as excessive protons, noxious temperatures (both hot and cold), mechanical pressure, and inflammatory mediators that are released from local immune cells. Activation of these membrane detectors leads to action potentials that carry the signal forward to indicate that a damaging event is ongoing which may threaten the organism. That signal propagates to the central terminals of sensory neurons and enters the central nervous system (CNS) through entry to the dorsal aspect of the gray matter of the spinal cord (known as the dorsal horn). Within the central terminals, the signal leads to the release of specific neurotransmitters (e.g. substance P, Calcitonin Gene Related Peptide (CGRP), and glutamate) that traverse the synapse and bind to a second set of detector ion channels and/or receptors on secondary spinal neurons. The transmission of the damage signal to the second neuron in the relay carries the message through the spinal column to the brain to a third neuronal relay. The message is subsequently delivered to multiple cortical areas where the signals are integrated and interpreted as pain. An adaptation to speed the response is provided by the spinal reflexes. In this case, a branch of the sensory neuron communicates the signal to interneurons, which may also receive some modulatory input from supraspinal centers. The integration of this input results in activation of motor neurons that enable the organism to remove itself from the damaging stimuli in order to stop the experience of pain. This is an essential evolutionary protective adaptation. Through focused application of stimuli to discrete regions containing sensory neurons, the study of the pain system has been enabled in a limited, controlled, and moderated manner. Rodents are the primary subjects included in such studies with occasional adaptations to larger species. Mostly the nerve endings intended for stimulation


reside in the epidermis of the skin, the plantar aspect of the hindpaw being the surface most frequently stimulated. Some variations include stimulation of the dorsal aspect of the hindpaw, forepaws, facial regions, and tail surface. Some models have been adapted to assess stimulation of visceral afferent sensory nerve endings particularly within the lower gastrointestinal tract. We know now from decades of neurophysiological and neuroanatomical studies that there are different populations of neurons that code for distinct forms of pain, such as mechanical, thermal, or inflammatory-like pains. Through application of discrete stimuli such as heat (Hargreaves et al., 1988; Janssen et al., 1963), pressure (Chaplan et al., 1994), chemical (Colburn et al., 2007; Sakurada et al., 1992), or laser activation (Tzabazis et al., 2005), the individual functions of these nerve populations are distinguished. The primary stimuli used to activate the epidermal nerve fibers in rodent are very frequently those that are also used in neurological exams (e.g. von Frey fiber monofilaments, Bryce et al., 2007; Haanpaa et al., 2011). The application of the stimuli tends to be of very short duration (seconds) as is the reflexive response. Through the reflex, the subject retains control of the stimulation as it is, in effect, escapable. The magnitude of the stimuli can be controlled or optimized in many cases through the range of temperatures, forces, concentrations of chemicals, duration of laser application, etc. In most cases, a range of intensity can be modulated and refined. Therefore, the focally restricted use of these optimizable stimuli effectively serves as a refinement approach.

3. Models of chronic pain Activation of the primary afferent nerve fibers through application of the stimuli described above is useful to probe the pain pathway, but in the absence of establishing a chronic pain state it does not fully reflect the condition of chronic pain. The initiation and the development of chronic pain results in significant alterations in membrane proteins, neurotransmitters and neuromodulators, neuroimmune mediators, and synaptic connectivity rendering the central and peripheral nervous systems under the state of the chronic pain significantly different from the normal state. These changes lower the thresholds at which external stimuli are perceived as noxious and the perception of pain magnified. These alterations can result in the suppression of some endogenous pain relieving systems and the enhancement of others. This knowledge has arisen from the neurophysiological and neuroanatomical evaluation of such systems in rodent models of chronic pain developed in the last several decades. Therefore, in order to understand the transmission of pain and the options for controlling ongoing chronic pain, these systems need to be assessed under the pathological condition. 3.1. Inflammation-based models The need to establish a true model for chronic pain in order to understand the biological changes associated with chronic pain was recognized as early as the 1970s (Sternbach, 1976). The first chronic pain models developed were based upon studies of inflammation following subcutaneous injection of various mycobacteria in rodents. It was hypothesized that inflammation established by introduction of the mycobacteria would evoke chronic pain associated with the inflamed tissue (Colpaert et al., 1980). It was further hypothesized that if subcutaneous mycobacterial injection was, in fact, hyperalgesic, subjects would pursue self-administration of oral analgesic drugs. Consistent with their hypothesis, the rats injected with complete Freund's adjuvant (CFA) consumed more from a bottle containing the non-steroidal anti-inflammatory drug (NSAID)/analgesic suprofen (Colpaert et al., 1980) or the opioid fentanyl (Colpaert et al., 1982) compared to a control solution. When the inflammation resolved, the analgesic self-administration diminished to control levels. Through

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using the self-administration of analgesics as an indirect indication of chronic pain, the individual subject acquires control of its own analgesic treatment. As the use of the inflammatory models expanded, adaptations were added to more closely mimic clinical inflammatory presentation. A variety of irritants (carrageenan (Hargreaves et al., 1988), capsaicin (Sluka, 1997), and CFA (Ren et al., 1992)) have been used to produce inflammation in various tissues to evoke an acute inflammatory pain associated with recruitment of neutrophils to the injected region, eliciting longer term pain responses associated with infiltrating macrophages (Gregory et al., 2013). Very often, the hypersensitivity that arises from inflammation has been probed using the aforementioned reflex measures. 3.2. Other etiology-specific chronic pain models It is known that various conditions of chronic pain are not biologically equivalent. Chronic pain is an umbrella term to describe a vast set of diverse sensory experiences with some common governing mechanisms and many significant distinctions. This complexity accounts for the expansion of the development of chronic pain models as a global refinement on the original inflammation-based models, in terms of tissues interrogated, chronic pain inducers applied, and measurements used. The goal has been to more closely mimic each specific human pain condition. There are many examples of such developed models over the last several decades. Through the study of these models it has been learned that the neurochemistry and the amenability to analgesic treatment can be very different, as it may be in clinic. For example, through establishment of models of neuropathic pain it was learned that the pharmacology of opioid analgesics under conditions of inflammation differ from that under the state of neuropathic pain. While opioids demonstrated enhanced analgesic potency in states of chronic inflammation (Millan et al., 1987; Neil et al., 1986; Stein et al., 1988), opioid analgesics under conditions of nerve injury show reduction in potency (Bian et al., 1995; Fairbanks et al., 2000; Mao et al., 1995; Nichols et al., 1995; Ossipov et al., 1997; Petraschka et al., 2007; Yaksh, 2002; Yaksh et al., 1995; Yamamoto and Sakashita, 1999) dependent on route of administration (Bian et al., 1995; Lee et al., 1995; Nichols et al., 1995) and ligand (Bian et al., 1995; Lee et al., 1995; Nichols et al., 1995). Therefore, the importance of development of models of chronic pain that reflect the specific pathologies of specific chronic pains has long been recognized. The next section features the development of seven key chronic pain models that were designed to mimic specific and distinct chronic pain conditions. This section is not intended to be a comprehensive survey of all the chronic pain models; for that we refer the reader to the excellent review by Gregory and colleagues (Gregory et al., 2013) and the recently published volume on various aspects of modeling chronic pain (Taylor and Finn, 2014). We hope through the next section to illustrate the differences in the chronic pain models and highlight how these differences are essential to the development of treatments specific to their individual mechanisms. 3.2.1. Peripheral nerve injury models During the last nearly thirty years a series of models of peripheral nerve injuries were introduced that have become the standards for studies of neuropathic pain. These involve partial nerve ligation (Seltzer et al., 1990), chronic loose constriction of the sciatic nerve (Bennett and Xie, 1988), tight ligation of the 5th and 6th lumbar spinal nerves (Kim and Chung, 1992), tight ligation and sectioning of the tibial and peroneal nerves (Decosterd and Woolf, 2000), among many adaptations and modifications. Although all nerve injury models require invasive surgery involving sectioning or ligation of nerve, the outcome of the injury is a chronic focal hypersensitivity of the dermatome associated

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specifically with that nerve. It has more recently been shown that subjects with chronic nerve injury-induced hypersensitivity, by and large, do not present overt signs of ongoing pain (Mogil et al., 2010) or display facial signs of pain as measured by the mouse grimace scale (Langford et al., 2010). In these models the hypersensitivity is detected primarily in the region innervated by the injury. The hypersensitivity that arises is associated with N-methyl-Daspartate (NMDA) receptor-dependent neuroplasticity in the spinal circuitry. This was recognized early on through demonstration that NMDA receptor antagonists (Mao et al., 1992), including the often used anesthetic ketamine (Mao et al., 1993), reduced the development of nerve-injury associated hyperalgesia. This is the reason why the use of ketamine is not recommended as an anesthetic in establishment of peripheral nerve injury-induced chronic pain models; instead, inhalation anesthestics are more commonly used. Through extensive neuropharmacological, neuroanatomical, and neurophysiological studies of the development and maintenance of neuropathic pain using these models, much has been learned and continues to be learned regarding alterations in analgesic pharmacological systems, spinal and supraspinal neural plasticity, the role of neuroimmune mediators, and identification of new neuromodulators. These advances could not have been achieved without the modeling conducted in rodents. 3.2.2. Cancer pain models Shortly after the development of the peripheral neuropathy models it was recognized that a method to model the unique aspects of cancer-evoked pain was needed. Pain is experienced by the majority of cancer patients due to tumor invasion and increases as the disease progresses; it is often difficult to control. It is intuitive that the interface of invading tumor cells with peripheral nerve endings establishes a unique biological environment distinct from that of the inflammatory milieu or the ensuing alterations from nerve injury. Therefore, information obtained from these preceding animal models is minimally informative for treating cancer pain. In the late 1990s and early 2000s, initial models of bone cancer pain were introduced. Osteolytic sarcoma cancer cell lines injected into the calcaneous (Wacnik et al., 2001) femur (Schwei et al., 1999), and humerus (Wacnik et al., 2003) bones resulted in enhanced mechanical and thermal reflexes, palpation-evoked pain, and movement-related pain by 21 days; all are symptoms reflective of the cancer patient experience. Since then, modifications of the model using distinct tumor cell lines have expanded analysis to other forms of cancer. These studies have enabled us to better model different types of cancers, to understand the impact of different types of cancers on bone (Lozano-Ondoua et al., 2013; Sabino et al., 2003), and to more closely represent the clinical experience. Neuropharmacological studies in these models have validated the effectiveness of opioid and alpha2 adrenergic agonists (Wacnik et al., 2000), cannabinoid receptor analgesic agonists (Kehl et al., 2003; Lozano-Ondoua et al., 2013), osteoprotegerin (Honore et al., 2000), and anti-nerve growth factor antibodies (Jimenez-Andrade et al., 2011). 3.2.3. Chemotherapeutic-induced neuropathy model In addition to the pain evoked from tumor nerve fiber interactions, a distinct but related painful neuropathy arises in cancer pain patients from commonly used chemotherapeutics. To model this condition, protocols were established to provide chronic exposures to commonly prescribed chemotherapeutics, such as paclitaxel (Polomano et al., 2001; Siau et al., 2006), taxol (Authier et al., 2000b), cisplatin (Authier et al., 2000a), vincristine (Siau et al., 2006), and oxaliplatin (Xiao et al., 2012). Following a course of repeated systemic injections, rodents typically develop peripheral hypersensitivity to mechanical stimuli and to cooling stimuli

within a week to fourteen days, similar to that observed in patients taking chemotherapeutics (Bennett, 2010). A notable and distinct response observed in subjects with nerve injury is a minimal to absent thermal hyperalgesia, which may illustrate differences in pathology (Bennett, 2010). Establishment of the chemotherapeutic neuropathy models have enabled identification of the specific pathology of the intraepidermal nerve fibers and have elucidated a specific axonal mitochondrial dysfunction (Bennett, 2010). Identification of this mechanism led to the evaluation of agents that enhance mitochondrial function (Jin et al., 2008; Xiao et al., 2009), which showed effectiveness in reversing chemotherapeuticinduced neuropathy. The great advantage of developing mechanism-based treatments to address chemotherapeutic-induced neuropathy is that, unlike typical nerve injuries, the exposure of a patient to these agents is predictable and, consequently, may be treatable with preventative drugs (Bennett, 2010).

3.2.4. Muscle pain models Musculoskeletal pain is a condition for which treatment is frequently sought. Musculoskeletal pain has diverse etiologies ranging from inflammation, non-inflammation based mediators, stress, and exercise. Therefore, a variety of muscle pain models have been established with the goal of mimicking these distinct clinical pain conditions. As mentioned above, much of the early work in pain modeling focused on cutaneous activation of sensory fibers. However, the innervation of cutaneous tissue is not equivalent to that of the muscle environment; C fiber afferent activation originating in muscle interacts differently (in a prolonged manner) from that of C fiber afferent activation in epidermal fibers. Therefore, the mechanisms cannot be extrapolated from models of pain focused on alterations in the cutaneous environment (Kehl et al., 2000; Radhakrishnan et al., 2003). The inflammatory agent, carrageenan, previously shown to result in cutaneous inflammation when injected in the intraplantar space, also evokes immunocyte infiltration when injected in muscle. Other similar models have injected carrageenan and other agents (e.g. mustard oil, CFA, and formalin) into various other muscles including orofacial muscles (Gregory et al., 2013). When carrageenan is unilaterally injected into the gastrocnemius muscle (Radhakrishnan et al., 2003) thermal and mechanical hypersensitivity are both induced on the ipsilateral hindpaw. When a higher concentration is injected (3%), the contralateral hindpaw becomes sensitized 1–2 weeks later, indicating mechanistic central nervous system alterations. When carrageenan is injected bilaterally intramuscularly in the triceps (Kehl et al., 2000) hypersensitivity is induced that can be detected by a reduction in movement-related activity; this effect is measured as a reduction in the force with which the forelimbs grip a mesh screen. This particular effect is reversed by delivery of analgesics, confirming that the reduction in movement is indicative of a pain state (Kehl et al., 2000). These protocols model myositis and muscle strains sustained by patients (Gregory et al., 2013). To induce non-inflammation-based muscle pain, several approaches have been developed. One extensively characterized model of repeated injections of intramuscular acidic saline (pH 4.0) into the gastrocnemius muscles results in a local intramuscular pH of 6.0–6.5 (a pH that activates muscle afferents (Steen et al., 1992)) and induces a persistent and widespread hypersensitivity to application of pressure (von Frey monofilaments, Sluka et al., 2001), reduction in general activity level, and alterations in the spinal cord dorsal horn neurochemistry (Gregory et al., 2013), all observations indicative of establishment of a chronic pain state. This model has been comprehensively assessed pharmacologically. Acid-induced muscle hypersensitivity is reversed by opioid agonists, NMDA receptor antagonists, and pregabalin, but not NSAIDS (Gregory et al., 2013).


In order to study mechanisms underlying the pain evoked by exercise and physical activity in chronic pain patients, models of exercise-induced muscle pain have been established. Several approaches are used. In one model, eccentric contractions are applied to the gastrocnemius muscle; in this case, the muscle of an anesthetized rat is electrically stimulated to contract repeatedly while simultaneously extended by rotating the associated foot (Alvarez et al., 2010; Proske and Morgan, 2001). This procedure results in significant muscle damage and a two hour mechanical hypersensitivity measured at the gastrocnemius muscle; it is intended to model delayed-onset muscle soreness (Gregory et al., 2013). A second model demonstrates that a prolonged period (2 h) of wheel running when combined with a low concentration of injected carrageenan produces cutaneous hypersensitivity (Sluka and Rasmussen, 2010). Just as the term chronic pain represents a broad spectrum of patient experience so muscle pain represents a spectrum of pain states arising in muscle tissue; distinct approaches for studying these mechanisms and screening potential treatments to address the specific mechanisms are needed. 3.2.5. Post-incisional pain model A model of post-incisional pain has been used for almost twenty years to study mechanisms underlying postoperative pain (Brennan et al., 1996). In this case, an incision is made in plantar skin of anesthetized rats with separation and retraction of associated muscle. This manipulation results in a thermal hypersensitivity of a five-day duration and a mechanical hypersensitivity of a five to ten-day duration, accompanied by central sensitization. Hypersensitivity evoked from mechanical (Stubhaug et al., 1997) and thermal (Martinez et al., 2007; Stubhaug et al., 1997) stimuli are similarly detected in postoperative patients and in a human model of incision pain (Kawamata et al., 2002). Spontaneous (non-evoked) behavior, such biting, licking or scratching of the hindpaw is evident post-surgery but does not persist beyond thirty minutes. Spontaneous activity of both primary afferent and dorsal horn neurons (the first and second neurons in the pain transmission relay) were increased on day 1 post-incision (Brennan, 2011). In a refinement of the model to more closely mimic the clinical condition in which muscle tissue (with distinct innervation relative to skin) is also included in the incision, the skin and associated deep muscle fascia are both incised and sutured. When muscle incision accompanies skin incision the spontaneous behavior of hindpaw guarding and spontaneous neuronal activity are both evident on day 1, and last at least five days (Xu and Brennan, 2009). It is proposed that the guarding behavior and spontaneous activity that accompanies this modified model of post-incisional pain may be more predictive of therapeutic agents to improve control of post-operative pain (Brennan, 2011). 3.2.6. Pancreatitis model The role of diet in various diseases is well recognized and some forms of pain are also associated with diet. Chronic alcohol (Warren and Murray, 2013) and a high fat diet (Lindberg, 2009) are exposures associated with development of pancreatitis in humans. Recently, a rat model based on high fat and chronic alcohol consumption was developed to mimic the pathology and the pain associated with human pancreatitis (Westlund and Vera-Portocarrero, 2012). Within three weeks, this diet induces a pancreatitis that presents pathological features comparable to that in humans. These include morphological changes and inflammatory infiltrates. Hypersensitivity to mechanical and thermal cutaneous stimuli develops in this model and continues for seven to ten weeks. Visceral hypersensitivity to application of von Frey monofilaments on the abdomen also arises and persists (Westlund and Vera-Portocarrero, 2012). Application of a herpes simplex virus (HSV) carrying a gene encoding pre-proenkephalin to the pancreatic tissue resulted in uptake of HSV-pre-proenkephalin

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virus into visceral sensory neurons coding for pain (Lu et al., 2007). This led to overexpression of the opioid peptide met-enkephalin in the pancreas and the region of the spinal cord dorsal horn associated with pancreatic input. Following overexpression of met-enkephalin, the thermal hypersensitivity typically evident in the model was reduced. Interestingly, signs of morphological changes (inflammatory infiltration, edema etc.) were also reduced (Lu et al., 2007). The insights gained from this model may lead to new, innovative gene therapy approaches designed specifically to treat this type of pain (Westlund, 2009). 3.2.7. Low back pain Very recently a model of low back pain has been characterized in a mouse line lacking a protein, Secreted Protein, Acidic, Rich in Cysteine (SPARC) (Millecamps et al., 2012, 2011). SPARC is normally expressed in the extracellular matrix of intervertebral discs, and decreased levels of SPARC have been reported in discs obtained from patients with intervertebral disc degeneration-related low back pain (Tajerian et al., 2011). Unlike most models, this is not an induced state of chronic pain but rather arises during the course of the animals’ natural life in the vivarium. In SPARC-null mice, the progression of degenerative disc disease begins at two months and is fully manifest by two years. The intervertebral disc degeneration and the associated signs of axial and radiating low back pain closely parallel that observed in patients (Millecamps et al., 2012). Unlike many pain models, these mice do not demonstrate cutaneous hypersensitivity to heat or mechanical stimuli. However, by nine months SPARC-null mice demonstrate withdrawal from stimuli such as acetone (Millecamps et al., 2011) cold water, and icilin (Millecamps et al., 2012), which activates neurons that code for cooling; this cold hypersensitivity is reversible with systemic morphine. Additionally, SPARC-null mice demonstrate reduced grip force indicative of movement-related hyperalgesia (described above in the muscle pain section). Motor performance on rotarod was equivalent between SPARC-null and normal mice, ruling out deficits in motor function. Finally, the SPARC-null mice demonstrate increased behavior directed at escaping the gravitational force on the spine inherent in the tail suspension assay (Millecamps et al., 2011) which has been shown to be reversed by systemic administration of morphine and clonidine given alone and in combination (Tajerian et al., 2012). These observations, taken over a significant period of time (415 months), have yielded a useful model of low back pain previously unavailable. Since this model is insensitive to the commonly applied sensory assessments described above, it illustrates that importance of careful sensory phenotyping. The introduction of this model will enable improved understanding of the anatomic and functional pathology of disc degeneration-related pain and will support the development of pharmacological and non-pharmacological therapies in a manner not previously available for low back pain.

4. Emerging assessments of pain: affect/motivational measures Finally, there is increasing consideration being given to refining pre-clinical assessment of chronic pain to include more affective or motivational aspects of the pain experience; for more discussion of these approaches the reader is referred to two recent, excellent reviews (King and Porreca, 2014; Murphy et al., 2014). In short, more complex behaviors are more frequently assessed in chronic pain models including, but not limited to, locomotor activity, depressed voluntary behaviors (Negus, 2013), facial expressions (Sotocinal et al., 2011), and motivation to pursue analgesics either through the conditioned place preference assay (King et al., 2009) or analgesic self-administration experiments (Gutierrez et al., 2011; Martin and Eisenach, 2001; Martin et al., 2007, 2006). Interpretation of the outcomes of these responses in chronic pain

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subjects becomes more challenging but may contribute to a more complete assessment of the chronic pain experience.

5. Local drug delivery: role in characterization of the pain and analgesia systems Identification of appropriate and effective analgesics and/or nonpharmacological treatments to control pain benefits from a mechanistic understanding of the source of the chronic sensory dysfunction. The development of largely restricted local delivery of drugs significantly advanced the definition of the neurotransmission/modulatory systems in the periphery, the spinal cord, and supraspinal sites that drive both pain transmission and inhibition of the signal (endogenous analgesia). These local drug delivery methods represent an important experimental refinement in that delivery of selective agonists and antagonists to a specific region enables molecular definition of key signaling intersections in a way not possible with systemic administration of drugs. In the case of systemic routes of administration, drugs are dispersed throughout the entire vasculature, including the CNS microvasculature and, therefore, effects of drugs cannot be ascribed with confidence to any particular region, even for those drugs with physiochemical characteristics permissive of crossing the blood brain barrier (BBB). Opportunities for the development of therapeutic interventions locally delivered specific points along the pain pathway have been identified and introduced; some of these are highlighted in the next few paragraphs.

5.1. Intraplantar drug delivery There has been significant interest in understanding the physiology of the peripheral nerve terminals, which are the specialized structures that receive information regarding sensation, including damaging stimuli indicative of pain. Some of the pain models involve direct activation of these structures through local intradermal hindpaw application of inflammatory agents, such as carrageenan, CFA, or capsaicin in the tissue containing peripheral nerve terminals (e.g. epidermis). Concurrently, ligands can be delivered intradermally in the affected hindpaw for assessment of their activity in the local environment (Katsuyama et al., 2013; Pereira de Avila et al., 2014; Sauer et al., 2014). Although, there may be drug redistribution through absorption to the systemic circulation, this confound can be addressed through limiting dose and through assessment of effects on the unaffected hindpaws. Such local delivery can greatly aid in distinguishing the mechanisms underlying peripheral nerve terminal sensitization and identifying effective therapeutic agents. Two notable approaches have arisen from mechanistic insight gained from the discovery and characterization of the TRPV1 receptor; the TRPV1 receptor resides in peripheral nerve terminals and is a transducer of pain signals initiated by either heat or the chemical capsaicin, the active ingredient in chili peppers; TRPV1 receptor agonists (e.g. topical capsaicin) and TRPV1 receptor antagonists have emerged concurrently as potential effective analgesic agents (Gunthorpe and Szallasi, 2008; Khairatkar-Joshi and Szallasi, 2009; Knotkova et al., 2008), capitalizing on distinct mechanisms. Activation of peripheral TRPV1 receptors by capsaicin results in a desensitization of the peripheral nerve terminals resulting in an analgesic effect; this is the mechanism by which topical capsaicin cream exerts its effect, with successful clinical application for diabetic neuropathic pain (Brederson et al., 2013). TRPV1 receptor antagonists have also been proposed for development to counter the initial activation of the ion channels by capsaicin and by heat. Clinical studies have had mixed results (Quiding et al., 2013).

5.2. Intrathecal drug delivery A common method employed to site specifically evaluate actions within the spinal cord is intrathecal delivery of drugs (Hylden and Wilcox, 1980; Mestre et al., 1994; Yaksh and Rudy, 1976b). Study of spinal cord signaling is, by nature, privileged for its relative separateness from the majority of the CNS. Therefore, the introduction of these CNS drug delivery approaches greatly opened up the possibility of specific characterization of the interaction between the first and second order neurons in the spinal cord. Based on the contemporary knowledge of the anatomy and vasculature of the spinal cord, a 19th century neurologist, Dr. James Corning, theorized that spinal delivery of drugs would result in localized anesthesia (Mackey, 1999). Dr. Corning pursued his idea by initially delivering cocaine solution intraspinally at the thoracic level in a dog. Consistent with his theory, this evoked 4 h hindlimb incoordination, weakness, and anesthesia. Based on that result, he then injected cocaine solution at the same spinal level in a human volunteer; by ten minutes the volunteer reported that his legs felt sleepy and he had impaired sensitivity to pinprick and electrical current, the duration of which was self-reported to be into the evening. This early translational study provided proof-ofconcept of the localized effect of the spinal drug delivery approach (Mackey, 1999). Much later, the development (Yaksh and Rudy, 1976b) and introduction of the chronic spinal catheterization model in rats was published in Science in 1976 (Yaksh and Rudy, 1976a), demonstrating continuous infusion of analgesics to the spinal cord (Yaksh and Rudy, 1976b). Within several years the first clinical trial was conducted and published (Wang et al., 1979), which laid the groundwork for what would become an effective approach for continuous localized delivery of analgesic medication to the spinal cord, where the first synapse of the pain pathway can be inhibited. The development and ongoing optimization of intrathecal drug delivery systems have enabled a subset of chronic pain patients, who are unresponsive to systemic treatment, to receive long term chronic spinal infusion of specific medications with physicochemical characteristics amenable to these systems (Ghafoor et al., 2007). Therefore, in addition to the neurochemical and neurophysiological information obtained from decades of localized spinal delivery to the first synapse (Yaksh, 2002), the rat model of chronic intrathecal infusion directly seeded the translation of this important option for some patients with severe pain. In 1980, a method for direct percutaneous intrathecal delivery by direct lumbar puncture in conscious mice was described and published in the European Journal of Pharmacology (Hylden and Wilcox, 1980); an adaptation for direct lumbar puncture in rat was subsequently described (Mestre et al., 1994). The application of these two intrathecal spinal delivery approaches (chronic catheterization (Yaksh and Rudy, 1976b) and direct lumbar puncture (Hylden and Wilcox, 1980)) have been extensively used and have greatly enabled our mechanistic understanding of the neurochemistry of the spinal cord sensory system. The first approach requires surgery to implant a catheter along the length of the spinal column, which enables chronic infusion and assures targeting of the infusate to the lumbar region. A notable refinement in this approach has been the introduction of a direct lumbar catheter implantation method (Storkson et al., 1996), the intention of which is implantation of the catheter at the target site. The second approach to intrathecal spinal delivery is direct lumbar puncture. This approach requires significant training and development of the injector to ensure accuracy of delivery (Fairbanks, 2003), but ultimately can be effectively applied in conscious rodents within an injection period on the order of seconds with little discomfort, analogous to the same delivery approach that is routinely given to


patients in the clinic (Harley et al., 2009). With experience and good training the risk for damage to the mouse spinal cord, infection, or immune responses is limited and/or manageable. The initial class of compounds evaluated spinally were the opioid medications, which were a logical selection as the effectiveness of the opioids as strong analgesics had been established for centuries. Further, the identification of the opioid receptors in the CNS had, at that time, recently been reported (Pert and Snyder, 1973). The effectiveness of the opioids as spinal analgesics was well predicted; the animal data corresponded well to the human clinical experience as we have now observed for the subsequent four decades. Additionally, the continuous spinal cord infusion model was used to assess the effectiveness of the alpha2 adrenergic receptor agonists (Sabbe et al., 1988) clonidine and dexmedetomidine (Takano and Yaksh, 1992), then known to have both anti-hypertensive and analgesic properties. Other classes of compounds demonstrating effectiveness in preclinical animal models when delivered intrathecally have included gabapentin (Hwang and Yaksh, 1997), cannabinoids (Fox et al., 2001), NSAIDs (Malmberg and Yaksh, 1992), NMDA receptor antagonists (Chaplan et al., 1997), and adenosine (Gomes et al., 1999) among others; these results have by and large corresponded to the human experience (Yaksh, 2002). The opioid receptor agonists are the most common analgesics delivered by the intrathecal route of administration in patients; other intrathecally delivered non-opioid agonists that have also shown effectiveness in human include the alpha2 adrenergic receptor agonist clonidine and A1 receptor agonist adenosine (Rauck et al., 2015). In addition to pre-clinical assessment of analgesic efficacy described here, the development of agents intended for intrathecal delivery require a distinct and essential additional form of animal modeling intended to assess neurotoxicity (Yaksh and Collins, 1989). These models include continuous infusion in larger animals in order to assess, for example, development of mass-occupying granulomas which are a concentration-dependent (Gradert et al., 2003) and ligand-dependent (Yaksh et al., 2013) outcome of some intrathecally infused agents. The large animal models closely parallel the human experience in terms of this adverse effect (Yaksh et al., 2002) and, therefore, represent a well-validated model for assessment of toxicity of agents intended for spinal infusion. Substantive review of this critical form of animal modeling is beyond the scope of this article but has been extensively detailed previously; for that the reader is referred to a series of articles by Eisenach et al. (2010), Eisenach and Yaksh (2002), and Yaksh et al. (2014). 5.3. Intracerebroventricular (ICV) drug delivery The delivery of agents to the intracerebroventricular space in mice has been used routinely, most often to evaluate the suprapsinal actions of a broad spectrum of analgesics. A method for drug delivery to the ventricular space in conscious mice was introduced in the fifties (Haley and Mccormick, 1957) and has been often applied for studies of supraspinal analgesia. This drug delivery approach has been particularly useful in delineating distinct actions of agonists or antagonists at supraspinal versus spinal sites of action (Bilsky et al., 1994; Lunzer and Portoghese, 2007; Pick et al., 1992) and to identify the circuitry mechanisms underlying the effects of analgesic combinations (Roerig, 2003; Roerig and Fujimoto, 1989). For continuous infusion of compounds in mice (Lenard and Roerig, 2005) or rats (Vonhof et al., 2003) cannula implantation via surgical procedures is necessary. The use of the ICV route of administration clinically is very limited; when accessed it is typically for delivery of opioid medication for palliative care (Raffa and Pergolizzi, 2012) in cases where systemically or spinally delivered opioids have become no longer effective. One case report (Lorenz et al., 2002) illustrated the effectiveness of substituting clonidine for morphine when ICV morphine became ineffective due to tolerance and/or disease progression. When

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morphine was reintroduced after a fourteen-day holiday, lower doses were effective when given in combination with ICV clonidine. This positive interaction is consistent with a significant pre-clinical literature demonstrating positive analgesic interactions from co-administered opioids and alpha2 adrenergic agonists (Fairbanks et al., 2009).

6. Correspondence of pain models to the human condition Recently, a much discussed publication (Begley and Ellis, 2012) featured the discordance of hematological and oncology models of cancer pain and the limited associated therapeutic development; there has been some discourse suggesting that rodent models of sensory dysfunction suffer similar limitations. This sentiment follows much discussion surrounding the disappointments of a widely discussed therapeutic, an NK1 receptor antagonist, that was effective in pre-clinical models of pain, but was not effective in clinical translation (Hill, 2000a). This prominent false positive example that emerged from pre-clinical pain research resulted in much discussion and consideration of the source of discordance, which ranged from testing approaches (Villanueva, 2000) and species differences (Urban and Fox, 2000) to clinical trial selection (Hill, 2000b; Laird, 2001; Urban and Fox, 2000). This experience has elevated awareness of many considerations in modeling chronic pain pre-clinically and the challenges for meaningful clinical translation of discoveries. It is also recognized that identification of false positive and false negative correspondence between pre-clinical screening and clinical efficacy trials of analgesics is, in fact, fairly limited, perhaps by the limited nature of publishing negative results (Rice et al., 2008). While the historic models may not predict translation of all systems, there remains significant and distinct correspondences to human clinical experiences that are also broadly acknowledged (Rice et al., 2008). In addition to the aforementioned agents, many new mechanism-based treatments have been shown to be effective both in pre-clinical models and corresponding patient populations. For example, the weak opioid receptor agonist and serotonin/norepinephrine reuptake inhibitor, tramadol, reduces manifestations of neuropathic pain in both rodent models (Hama and Sagen, 2007; Millecamps et al., 2011) and human patients (Sindrup et al., 1999), as has gabapentin (Abdi et al., 1998; Backonja, 2000; Hwang and Yaksh, 1997). Etanercept, the tumor necrosis factor (TNF) inhibitor, has proven an effective treatment in patients with rheumatoid arthritis as well as CFA-treated rats (a model of rheumatoid arthritis) (Inglis et al., 2005). Sumatriptan has been very widely known to be an effective and selective treatment for migraine pain in humans; similarly, relatively newly developed rodent models for migraine have demonstrated effectiveness of sumatriptan for reversal of hypersensitivityrelated (Bates et al., 2010; Pradhan et al., 2014) and neurochemical (De Felice et al., 2013) responses. Intrathecally delivered ziconitide (SNX-111), an N-type calcium channel blocker, reduces thermal hypersensitivity in nerve-injured rats (Scott et al., 2002) and provides analgesic relief to cancer and herpes simplex virus (HIV) patients (many with peripheral neuropathy) (Staats et al., 2004). Additionally, the introduction of the spinal catheterization method described above (Yaksh and Rudy, 1976a), led to an important treatment option for many patients for whom systemic opioid medication is not effective (Ghafoor et al., 2007). While the correspondent rodent to human pharmacology is not intended to be a comprehensive list, it does illustrate similar pharmacological outcomes within pain models that compared to the human pain condition. Recognition of the correspondent pharmacology is not to discount appropriate concerns of false negatives and false positives that may arise from pre-clinical modeling. The basic pain and clinical research community are cognizant of the limitations of the animal models (Rice et al., 2008). To address such limitations an emphasis has emerged on

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identification of ongoing refinements, such as have been featured in this article, to more closely mimic specific pain conditions intended for either further study of mechanism and/or development of new treatments. An additional focus has been on ensuring that experimental standards are used to eliminate user bias (e.g. blinding and experimental replication). A third major drive has been to expand the outcome measures to include assessments that more fully assess the affective aspects of pain (King and Porreca, 2014; King et al., 2009) as previously mentioned. Going forward, the broad recognition that these further refinements to pre-clinical pain research strategies are needed will likely yield additional treatment options at various intervention points along the pain pathway; these therapeutic strategies are so greatly needed globally. To paraphrase Finding 5-1 from the IOM report on pain (IOM, 2011), much has been learned regarding the understanding the basic mechanisms of nociception and pain, yet much remains to be to be learned. With the aforementioned global recognition of the great need for comprehensive and effective pain management, the advocacy for increased research on pain, and the recent development of the pathology-specific chronic pain models, anticipation for new and effective therapeutics is very high. There remains a critical need for continued support of basic and clinical research on pain.

7. Pain and analgesia research and the 3Rs As members of the research compliance community, Institutional Animal Care and Use Committee (IACUC) members and principal investigators of chronic pain protocols should consider the principles introduced in 1959 by Russell and Burch (Russell and Burch, 1959) and embodied as the “Framework of the 3Rs:” Replacement, Reduction, and Refinement. In the decades that followed, these terms have been promoted to become the standard by which investigators are evaluated for their consideration of animal use in their research and IACUCs are, in turn, evaluated for their review of protocols. Legislation has been enacted in some countries to assure compliance with these expectations and funding agencies and accreditation agencies may exact such compliance through their guidelines. In order to be responsive principal investigators should describe their consideration of all 3Rs and diligent IACUC members should review chronic pain protocols with consideration for all three aspects of the framework. Given the 3Rs emphasis, it is important to consider the opportunities and limitations for application as they relate to pain research. 7.1. Replacement It is suggested that the replacement arm of the 3Rs framework can be accomplished through a variety of approaches such as tissue culture, perfused organs, tissue slices, cellular fractions, subcellular fractions, in silico simulations, human volunteers, and meta-analyses. However, the global objective of fully characterizing the mammalian pain and analgesic systems within the central and peripheral nervous systems and understanding its development in the whole organism requires intact systems. While much can be learned about basic neurophysiology using in vitro models of cultured peripheral sensory neurons or CNS cells in a simpler and more controlled media environment by removing them from their normal environment, these cells are likely to display properties that are not truly reflective of the natural state. Phenotypic (Araki et al., 1988; Ferro et al., 1995; Nishi and Willard, 1988; Rao and Landis, 1993) and electrophysiological (Buchhalter and Dichter, 1991; Nishi and Willard, 1988; Turrigiano et al., 1995) differences between neurons in vitro and the same neurons in in vivo tissue have been observed. Therefore, since we currently have a very limited

understanding of the pain systems in intact tissue, in vitro systems cannot fully represent the complex in vivo environment. Computer models of the CNS are under intense development. These advances may, in the future, substantially enhance our ability to ask questions that will assist experimentalists in refining hypotheses and experimental protocols. A large fraction of major developments in this field have focused on modeling electrical events in single neurons (De Schutter, 1998, 1999; Gundappa-Sulur et al., 1999; Jaeger et al., 1997; Nelson and Bower, 1990). Modeling of realistic networks of CNS neurons commenced in the mid-nineties (Lukashin et al., 1996; Lukashin et al., 1994) and has focused largely on motor systems (Georgopoulos, 2014). Current modeling of sensory circuitry in the spinal dorsal horn (Le Franc and Le Masson, 2010; Zhang et al., 2014) appears to be limited thus far to acute behavior, not incorporating the maladaptive synaptic plasticity that underlies chronic pain. The complexity of the sensory system suggests that use of computer models to study chronic pain will be of limited value for the foreseeable future. While Replacement of animals in our ongoing quest to understand the pain pathways and analgesic systems is not a realistic goal (on a physiological, anatomical, or a molecular level), it is important to consider preliminary work that can be and is often done in the initial phases of basic research. These phases do include cell based or ex vivo tissue systems to screen for potential targets, confirm mechanisms of action, etc. However, in order to study the experience of pain, an intact mammalian system that reflects the complete experience (including cognitive function) is needed. 7.2. Reduction With respect to Reduction, there are sometimes opportunities within experimental designs to potentially reduce numbers or to organize experiments to minimize repeated control groups. Investigators are advised to do so when possible; in addition to the importance of consideration of Reduction, economic realities are also consistently persuasive in this direction. However, it is not always practical or appropriate to organize experiments to minimize repeated control groups. Control groups are an essential aspect of scientific inquiry; these groups ensure monitoring effects of environmental fluctuation (Rice et al., 2008) on experimental outcomes, for example. Therefore, it must also be acknowledged that insufficient consideration of the impact of Reduction of animals on the quality of experimental design can result in outcomes that compromise the data. Included in these risks are the underpowering of studies, failure to replicate work, or failure to appropriately replicate the work of others. All of these failures could render the value of the data collected from remaining subjects limited or even unusable. One criticism often levied at basic research is the reported failure of many investigative groups to adequately replicate their findings (Begley and Ellis, 2012) prior to publication. The impact of this is publication of potentially false positives that can lead other groups down false trails, needlessly using more animals than what would have been expended in the replication. The basic principles of reproducibility and ensuring robust control should not be compromised in the interest of “acute” reduction of animals. The consequence may, in fact, yield an overall greater consumption of animals to limited useful purpose. With respect to this scenario the old saying “penny-wise and pound-foolish” comes to mind. Another common approach for reducing the number of animals requested/used is to reuse an individual subject in experiments for which the subject would not otherwise have been used, thus eliminating the need to request additional animals for the new set of experiments. There are often situations where re-using a subject is appropriate and desirable, particularly when the prior experimentation was relatively non-invasive, such as a reflex measurement of very minimal duration; under such conditions, re-use may meet the spirit of the Reduction arm of the 3Rs.


Regarding this matter, the Guide for the Care and Use of Laboratory Animals indicates that “Principal Investigators are strongly discouraged from advocating animal reuse as a reduction strategy, and reduction should not be a rationale for reusing an animal or animals that have already undergone experimental procedures especially if the well-being of the animals would be compromised (Carbone, 2012).” The latter condition is certainly always true: the well being of the individual animal must be assessed prior to the conduct of all procedures. However, in the case where the subject has completed its primary test or experiment, has no evidence of lack of well-being or normal function, and is considered appropriate for additional experimentation, it can also be debated whether continuing that particular subject's term as an experimental subject contributes to the goal of optimizing animal welfare. Certainly, from the perspective of the individual subject, the burden of experimentation may merely have shifted to double the experience of the individual subject in the name of reducing overall animal numbers. This seems counterintuitive to an optimal animal welfare environment; each investigator and IACUC reviewer should consider such matters on a case-by-case basis when evaluating the response to the Reduction aspect of the 3Rs and use objective, sound professional judgment. A recent publication (Balcombe et al., 2013) claimed that pain research in general fails to consider the 3Rs, with a primary emphasis on a perceived lack of attention to the Reduction arm. However, the premise upon which this was based was seriously flawed. First, the authors searched in 55 pain research papers (uncited) for the words Replace, Reduce, Refinement. These terms were not evident in any of the studies surveyed. The authors suggest that omission of these terms infers lack of knowledge of or an indifference to the 3Rs framework on the part of the investigators. However, it is widely known that it is not conventional to include such terms in scientific publications. Rather, these terms would have been included and addressed in the IACUC-approved protocols associated with the work represented in the 55 publications (uncited). The second parameter featured was an elevation in pain research using animal models over recent decades (Balcombe et al., 2013). Since the authors focused on models of chronic pain from 2000 to 2010, such an observation is to be expected. During the 2000s, transgenic models were introduced enabling mechanistic evaluation not previously possible. Also, as noted previously, the declaration of the Decade of Pain Control and Research (2001– 2010) shed further light on the need for focused research in pain mechanisms. As described earlier in this review, new pain models continue to emerge to more closely mimic the specific pain conditions observed in the clinic. Therefore, it is not surprising to observe in increase pain research over the last decades. Finally, regarding the assertion of Balcombe et al., of a lack of animal welfare consideration in the pain research community, it is important to consider the limited basis for such a claim in the context of an international scientific effort to understand a globally recognized therapeutic public health problem.

7.3. Refinement The traditional concept of refinement is to minimize pain and distress where possible. For pain research, this is also the case, but it is more challenging because the sensory experience of pain is, in fact, the biomedical condition that requires investigation in order to develop therapeutic treatments. In the case of pain research, refinements can, however, be achieved through attempting to restrict the pain to a localized region or specific tissue when appropriate and possible or to focus upon specific chronic pain syndrome of study. Refinement can also be achieved through consideration of earliest endpoints.

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Throughout this review, we have described the optimization of chronic pain models to most closely mimic the human pain condition as well as specific and substantive refinements, which improve the outcomes of the research. Consequently, the experience for individual subjects is improved and, ultimately, the total number of subjects needed to acquire the knowledge sought is reduced. These refinements may represent the greatest potential impact of the 3Rs for research on pain and analgesia mechanisms. These refinements are organized along a progression of pain modeling from the simplest study of reflexes to the more complex characterization of the complete pain experience. For example, where possible, the use of escapable reflex measures confines the experimental period of stress or pain to a few seconds and ensures that the subject controls the experiment by signaling the termination of the experiment through reflexive response. However, sole reliance on reflex measures risks failing to capture the complete experience of pain and, likely, fails to detect important therapeutic opportunities. As stated earlier in this review, it is widely recognized that chronic pain arises from distinct injuries, exposures, and pathologies with some overlapping and some distinct mechanisms of action. Therefore, additional models of chronic pain are required to address these distinct pain conditions. The development of the cancer pain models (Schwei et al., 1999; Sluka et al., 2005; Wacnik et al., 2001) yielded much mechanistic information; however, unlike the neuropathic models, the outcome of introduction of oncogenic tumor cells have presented symptoms of ongoing spontaneous pain and evidence of movement-evoked pain (Wacnik et al., 2005). These outcomes are important because they mirror the disease condition that they are intended to model. However, refinements continue to be incorporated that improve both the match to the human condition and the experience for the research subjects. For example, the original method for delivery of osteolytic sarcoma tumor cells to bone was subsequently refined in order to restrict the tumor to the intramedullary space of bone (Sabino et al., 2003), a refinement that led to a model more closely reflective of primary or metastatic bone cancer. Further, evaluation of additional tumor cell lines further optimized the model (Sabino et al., 2003), to more closely reflect the heterogeneity inherent in clinical bone cancer pain. In all cases, the time course for progression of disease is much shorter than that of the neuropathic pain models and so the endpoints are early and the monitoring of disease progression is more frequent. A general refinement that has arisen recently is the move toward inclusion of operant measures as an indication of ongoing pain and, in particular, pain relief. Under these experimental conditions the subjects act on the opportunities for analgesic drug administration. In fact, the very first modeling of chronic pain included an operant measure to assess the self-administration of an NSAID, suprofen (Colpaert et al., 1980), and fentanyl (Colpaert et al., 1982), as consumed by rats with chronic inflammation. Similar approaches have been more recently applied to assess both opioid (Colpaert et al., 2001; Martin et al., 2007; Wade et al., 2013) and non-opioid (Gutierrez et al., 2011; He et al., 2012; King et al., 2009; Martin et al., 2006) forms of self-administration or analgesic drug-related conditioned place preference. These experimental designs are, by nature, more complex but provide an opportunity to more closely model an aspect of pain management, patientcontrolled analgesia. What we hope is evident from the summary presented above related to a broad spectrum of chronic pain models is that Refinement is an ongoing part of experimental design to approach as closely as possible the corresponding broad spectrum of complex human clinical pain conditions. At the forefront of Refinement remains the goal to minimize the experience for the individual research subject to the greatest extent possible,

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especially in identifying the earliest endpoints that can be used, while maintaining the integrity of the scientific objectives.

F. Kitto whose devotion to the well-being and caring treatment of our research subjects embodies the value of Refinement.

8. Conclusion References Ongoing optimization of methods to mimic the human condition and to minimize pain and distress for research subjects through refinements, the measurements that we use, and by identifying early endpoints when possible is, ultimately, the best practice. Additionally, it is essential to focus on training and communication within research groups to ensure optimum experimental design and implementation practices and thus, the best outcomes both from a scientific and animal welfare perspective. In fact, problems that arise in the research setting are very often due to miscommunication or inadequate training rather than poor intentions. The need for increased training of researchers experienced in pain research techniques has been recognized and appears as Recommendation 5-5 in the IOM report (IOM, 2011). Experimental design and acquisition of the techniques to appropriately and humanely engage in pain research does require direct training from experienced principal investigators. Early training of research trainees and, in fact, all team members should include competency in the principles of animal welfare, the 3Rs, and simple gentle handling; such concerted engagement of all trainees and team members in the investigator–veterinarian–IACUC partnership or three-legged stool will greatly elevate the research enterprise and improve animal welfare. IACUC reviewers of chronic pain protocols can likewise simultaneously ensure and improve animal welfare and advance the pain research effort through keeping a number of considerations in mind through their reviews and oversight of chronic pain research. These include consideration of each pain research protocol in the context of the larger scientific question/goal, application of objective and informed consideration as to where the 3Rs can and should (or should not) be applied, and evaluation and assurance that the principal investigator and his/her team members have appropriate training in the specific methods related to pain research. It is important for the IACUC, in general, to engage investigators in a strong partnership to facilitate the research mission of the institution while maintaining the animal welfare interests of the research subjects. These are mutually synergistic and achievable goals. For institutions that include research on chronic pain within their portfolio, it is important to include pain research experts in the IACUC membership/leadership and animal welfare leadership; such an approach capitalizes on such expertise, encourages a context-driven approach to evaluation of the 3Rs, and engages the pain research investigators in the assurance of animal welfare. Institutions that have taken this approach have made significant progress in fostering important bidirectional communication through engagement of the investigators and their team in the animal welfare mission; the outcome has been improved application of science and improved environments and experience for the research subjects. Such approaches will optimize the experience for the research subjects as well as ensure that our global objective to substantially improve pain management will be realized.

Acknowledgments We would like to express our appreciation to Drs. Henk-Jan Schuurman for the invitation to contribute this review, Drs. George Wilcox, Lucy Vulchanova, Laura Stone, and Carrie Wade, and Kathleen Sluka for comments on this manuscript. We would like to especially thank our primary research team member, Mr. Kelley

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