REVIEW ARTICLE

Internal and External Factors Affecting the Development of Neuropathic Pain in Rodents. Is It All About Pain?
K. Vissers, MD*; R. De Jongh, MD, PhD*; V. Hoffmann, MD; R. Heylen, MD, PhD*; B. Crul, MD, PhD**; T. Meert, PhD, PhD
*Multidisciplinary Pain Center, Ziekenhuis Oost-Limburg, Genk, Belgium; Department of Anaesthesiology and Multidisciplinary Pain Center, University Hospital Antwerp, Edegem, Belgium; **The Pain Center, Department of Anaesthesiology, University Medical Center St. Radboud, Nijmegen, The Netherlands; J&J PRD, Turnhoutsesteenweg 30, Beerse, Belgium

Abstract: It is important to know the factors that will inuence animal models of neuropathic pain. A good reproducibility and predictability in different strains of animals for a given test increases the clinical relevance and possible targeting. An obligatory requirement for enabling comparisons of results of different origin is a meticulous denition of the specic sensitivities of a model for neuropathic pain and a description of the test conditions. Factors inuencing neuropathic pain behavior can be subdivided in external and internal factors. The most important external factors are; timing of the measurement of pain after induction of neuropathy, circadian rhythms, seasonal inuences, air humidity, inuence of order of testing, diet, social variables, housing and manipulation, cage density, sexual activity, external stress factors, and inuences of the experimenter. The internal factors are related to the type of animal, its genetic background, gender, age, and the presence of homeostatic adaptation mechanisms to specic situations or stress. In practice, the behavioral presentations to pain depend on the combination of genetic and environ-

Address correspondence and reprint requests to: Kris Vissers, MD, Multidisciplinary Pain Center, Ziekenhuis Oost-Limburg, Campus Andr Dumont, Stalenstraat 2, 3600 Genk, Belgium. Tel: +32 89 32 52 40; Fax: +32 89 32 79 11; E-mail: kris.vissers@skynet.be. 2003 World Institute of Pain, 1530-7085/03/$15.00 Pain Practice, Volume 3, Issue 4, 2003 326342

mental factors such as accepted social behavior. It also depends on the use of genetic manipulation of the animals such as in transgenic animals. These make the interpretation of data even more difcult. Differences of pain behavior between in- and outbred animals will be better understood by using modern analysis techniques. Substrains of animals with a high likelihood for developing neuropathic pain make the unraveling of specic pathophysiolocal mechanisms possible. Concerning the effect of stress on pain, it is important to differentiate between external and internal stress such as social coping behavior. The individual dealing with this stress is species sensitive, and depends on the genotype and the social learning. In the future, histo-immunological and genetic analysis will highlight similarities of the different pathophysiological mechanisms of pain between different species and human subjects. The nal objective for the study of pain is to describe the genetics of the eliciting pain mechanisms in humans and to look for correlations with the knowledge from basic research. Therefore, it is necessary to know the genetic evolution of the different mechanisms in chronic pain. In order to be able to control the clinical predictability of a putative treatment the evolutionary pharmacogenomic structure of specic transmitters and receptors must be claried. Key Words: genetics, neuropathic pain, environmental, species, strain

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INTRODUCTION
Neuropathic pain is characterized by spontaneous and/or abnormal stimulus-evoked pain, dened as allodynia when caused by normally innocuous stimuli. Its treatment represents a major challenge due to its clinical complexity and the lack of adequate drug treatments.1 Because experimental conditions in humans are unethical several animal models were introduced in order to study neuropathic pain.26 The majority of these models are based on the introduction of functional disturbances within peripheral nerves, resulting in increased responsiveness to well-dened innocuous or painful mechanical, thermal, and chemical stimuli.2 Within clinical practice, however, complaints of a patient confronted with neuropathic pain are often based on different nociceptive inputs.7 The clinical response to nerve injury and to the different treatment modalities can vary among patients with comparable pain syndromes.8 Surprisingly, not all patients will present with a neuropathic pain behavior after a demonstrable nerve injury.9

To illustrate the complexity of the study of pain, in the different animal models used nowadays, variations in neuropathic pain behavior and pharmacological responses are described.1013 These variations may be attributed to internal and external inuencing factors.14 See Figure 1. Internal factors include strain and species specicity, gender, age and stress reactivity. External factors known to inuence pain behavior are: timing of the measurement of pain post induction of neuropathy, circadian rhythms, inuence of season and humidity, inuence of order of testing, diet, social variables, housing and manipulation, cage density, sexual activity, external stress factors, and an inuence of the experimenter. The knowledge of these factors and their impact on the experimental conditions is crucial to enhance the reproducibility of these tests. This will improve the accuracy of the interpretation of the results and allows a better correlation with clinical situations. The increasing interest in clinically relevant animal models for the evaluation of pain and the genetic sensitivity to nociception make studies on genetically manipulated animals important.15,16 This review will focus on the

Factors influencing neuropathic pain in rodents

Internal factors

External factors
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Diet

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Polymorphism

G E N E T I C

Circadian rythms Species / strain / Substrain Social variables Transgenic animals Stress factors Knock-out Sexual activity Gender / Age

Season and humidity Timing of the experiment

I R O N M

Influence of experimenter Housing and manipulation Order of testing

E N T A L

Figure 1. Schematic overview of the internal and external factors inuencing neuropathic pain behavior in rodents (as described in the text).

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description of different factors inuencing the neuropathic pain behavior in animal models.

BASIC KNOWLEDGE TO STUDY NEUROPATHIC PAIN

A denition of what constitutes neuropathic pain is necessary for guiding explanatory basic research or establishing the clinical prognosis, and is important for therapeutic approaches and design of treatment trials of novel analgesic drugs that are currently under development. The International Association for the Study of Pain denes neuropathic pain as pain that is initiated or caused by a primary lesion or dysfunction or transitory perturbation in the peripheral or central nervous system.17 Characteristics of neuropathic pain are an increased responsiveness to different innocuous and painful mechanical, thermal, or chemical stimuli.1 Neuropathic pain may be the result of a lesion of the peripheral or central nervous system. All types of neuropathic pain are projected to the innervation territory of the damaged nerve or pathway according to the somatotopic organization of the primary somatosensory cortex.18 In clinical conditions, however, damage to the nervous system is not always demonstrated. Neuropathic pain symptoms do not always share the same fundamental mechanisms and a standard analgesic treatment may have different responses in a specic condition.19,20 Not all patients will present neuropathic pain behavior after a demonstrable nerve injury.9 Animal Models for the Study of Neuropathic Pain In animals, the study of pain and its control is an important area of basic biomedical research, assembling a number of signicant advances in the understanding and the treatment of acute and chronic pain in humans. Animal models are preludes to clinical trials, not substitutes. Animal models have to be validated, for example by showing responsiveness to treatment modalities effective in humans. Behavioral studies of pain in animals contribute essential information on clinical pain conditions, which cannot be obtained in any other way. Nevertheless, the specic expression of discomfort can be misinterpreted as pain behavior. They express behavioral responses to noxious stimuli such as: withdrawal, escape, defense/attack postures and movements, alarm and distress vocalization, writhing, autonomic and endocrine responses, protective postures, mouthing of the affected limb, sleep fragmentation and avoidance

learning21. The study of normal and abnormal behavior requires the study of nociceptive behaviors. Animals can be bred to become standardized in terms of genetic and behavioral conditions, which makes them ideal study objects. Humans, biologically seen, can be compared to animals, they are however subject to important non-genetic inuences, which makes them more difcult for analytical comparisons22. Some of the disease states associated with neuropathies in humans can be modelled in animals. Most of these models are based on peripheral nerve injury. For reasons of reproducibility and simplicity, most studies of neuropathic pain use traumatic nerve injury, usually in rodents22. It becomes clear that the different models share a common feature of degeneration of some, but not all, sensory bers in a major peripheral nerve23. Experimental neuropathic pain in animals: (See gure 2) The rst model of neuropathic pain was developed by Wall in 1979 and consisted of a complete transsection of the sciatic nerve of the rat, followed by either tightly ligating the proximal stump or placing it into a sealed tube6. This model was modied in a neuroma model and an axotomy model24. More recently developed models for neuropathic pain rely on more partial denervation. A widely accepted model is the chronic constriction injury model (CCI) described by Bennett and Xie, which loosely constrict the rats sciatic nerve. This is considered as a peripheral mononeuropathic

A
L3 L4 L5 L6 S1

3 2 1 C

Figure 2. Schematic representation of the spinal cord (A), the exiting spinal nerves (B) and the sciatic nerve (C) with the specic anatomical locations of nerve injury applicated in different models of neuropathic pain in rodents: (1) axotomy model (Wall)6 (2) Bennett model (CCI)2 (3) Seltzer model (PSNL)5 (4) Chung model (SNL)3 ( represents a transection of the nerve; I represents a ligation of the nerve).

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animal model2. The partial sciatic nerve ligation model (PSNL) was proposed by Seltzer et al. where the sciatic nerve was partially, tightly ligated at the periphery5. There are both similarities and differences in the behavioral syndrome following both types of lesion. First, whereas the allodynia and hyperalgesia disappear after 4 to 6 weeks in the CCI model, mechanical allodynia in the PSNL model was reported to exist for more than 3 months. Second, allodynia to cold stimulation is only observed in the CCI model and not in the PSNL model12. Prominent contralateral inuences of the mechanical and thermal allodynia are described in both models12,25. Hyperalgesia to heat stimulation exists in both models. Kim and Chung described the segmental nerve ligation (SNL) model, tying completely the L5 and L6 spinal nerves3. The behavior following this type of lesion much resembles that observed in the PSNL model. Dorsal rhizotomy and cryo neurolysis (CNL) have also been used as well as the exposure of the nerve to a laser beam described by Gazelius26,27,28. It is unlikely that a single animal pain model will activate the full range of neuropathic pain mechanisms29. In animals, where communication is difcult, we are committed to the assessment of the pain behavior generated by characteristic stimuli. Several pitfalls deserve attention during designing or interpreting the above-mentioned neuropathic pain animal studies. At the level of the spinal cord, it is questioned whether a manipulation serves to alter the observed behavior, because it actually makes the pain state more or less intense, or because it is possible that it produces competing behaviors. The absence of response, in the face of a stressor, is not necessarily an indication that the animal is without sensation. Failure to observe a response in animals with lower motor neuron hyperpolarization and unable to move is not an indication of analgesia. Studies with a2 adrenoreceptor agonists indicate that stress induced by immobilization and immobilization with long-term-applied paw pressure unmasked possible antinociceptive properties of the various a2 adrenoreceptor agonists30. Physiology of Neuropathic Pain Models Damaged Sensory Fibers. Wallerian degeneration within endoneurial nerve bers is the principal pathological nding in the generation of neuropathic pain behaviors in most animal models and is dependent on activity of macrophages recruited to the injury site.31

Ramer et al. reported that Wallerian degeneration is required for subsequent sympathetic sprouting into the DRG.32 Sympathetic sprouting follows the induction of neuropathic pain in most models of neuropathic pain.33 However, this sympathetic growth is rather slow after a nerve lesion. This sympathetic-sensory coupling can explain the variation in the presentation of neuropathic pain. Until now, it is not yet clear why some chronic pains are dependent on an intact sympathetic nervous system, while others are sympathetically independent.34 In neuropathic pain conditions, axotomized afferent neurons begin to discharge spontaneously. This afferent barrage provides constant input in the CNS, and may induce central sensitization.35 The damaged sensory neurons show changes in gene expression that affect virtually all aspects of the neurons function. Different neurotransmitters or neuromodulators are produced by the damaged afferents and released in the spinal cord. Some damaged A bers and different nerve types appear to undergo a phenotypic shift and begin to express transmitters normally associated with nociceptors, ie, substance P and brain-derived neurotrophic factor (BDNF). There is good evidence that these factors are important contributors to central sensitization.36 Moreover, damage to some afferents in a peripheral nerve leaves the remaining, intact, neighboring bers facing less competition for target-derived factors, such as substance P, excitatory amino acids (EAAs), prostaglandins and NO, and subject to putative degeneration factors in the peripheral nerve. Spared Sensory Neurons. Spared sensory neurons do not undergo Wallerian degeneration, and are not subject to reduced access to target-derived trophic factors, such as substance P, EAAs, prostaglandins, and NO. Spared afferent bers frequently show the opposite pattern of gene expression to that seen in damaged axons and many of the examples of altered gene expression in this group can most parsimoniously be explained by increased availability of nerve growth factor (NGF).37 The molecular basis for the increased ectopic activity in spared afferents is not well understood.35 Intact spared afferents show remarkable plastic changes including the development of spontaneous activity.35 The discharges in myelinated bers (overwhelmingly innocuous mechanoreceptors originally) may only produce pain if they impinge on a CNS sensitized by the nociceptor inputs.35 Whether these spared sensorial bers and

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C-bers are species specic and genetically inuenced is unknown. Assessment of Neuropathic Pain More research is needed to evaluate whether a certain test in an animal is the most optimal one. The behavioral tests that are used to study nociception nociceptive testsevaluate input-output systems that function via black boxes which the neurobiologist wishes to decode. When describing these tests, it is important to specify the characteristics of the input (the stimulus applied by the scientist) and the output (the reaction of the animal).22 The inputs and outputs of these systems are very intimately linked by the physical characteristics, notably the temporal nature, of the stimulus. In humans and in animals, experimental studies of the mechanisms underlying acute pain necessitate the use of appropriate stimuli to provoke the desired sensations. To be adequate, these stimuli have to be quantiable, reproducible and noninvasive.38 Spontaneous Pain. It is difcult to detect spontaneous pain in animals. It is even more difcult when allodynia is present. Therefore the analysis of spontaneous pain behavior can be reported during test conditions but is mostly not used in the nal interpretation of data obtained from test experiments.2 Possible signs of this spontaneous pain behavior are lifting or shaking the concerned hind paw, licking this hind paw or holding it in a guarded position. More specic spontaneous pain behavior is the increased incidence of autotomy, where animals scratch and bite their denervated and anesthetic limb. This may be mild or severe and originates from the spontaneous activity from injured nerves.39 More general signs of spontaneous pain behavior include differences in sleep patterns, exploratory behavior, nutritional status and social behavior. Mechanical Allodynia. One of the most frequently used tests for mechanical allodynia is the Von-Frey test applying gradually increasing pressures with monolaments on the paw or tail.40 This test is commonly performed with the animals restrained under a plastic cover. The response to mechanical probing with a xedforce Von-Frey lament depends on such details as the kinetics of force application, the condition of the lament tip such as a sharp spur. Even the choice of housing cage affects cutaneous sensitivity.41 The automated Von-Frey test as described by Meert is measuring the

withdrawal threshold of the tested hind paw by using an electronically regulated increasing mechanical stimulation.42 Thermal Allodynia. Thermal allodynia and hyperalgesia can be measured by placing the animal on a metal plate that is either heated or cooled to a stable temperature (abnormal for the given species) and measuring the latency time for appearance of typical pain behavior. The animals respond to this stimulus by lifting and licking or biting the paw.43 The cold plate was found to be a reliable method for detecting abnormal nociceptive behavior even at long intervals after nerve or inammatory injuries, when responses to other nociceptive stimuli have returned to near normal, as is the case in heat allodynia.44 Moreover, a primary advantage of the cold plate test is that it allows observation of freely moving animals in a minimal stress environment. It allows the use of a wide range of temperatures, from noxious cold to neutral temperature; the animals can be tested on different time schedules or durations of exposure. In contrast the acetone test in which a droplet of acetone is applicated to the plantar surface of the paw, does not permit various duration of exposure since a brisk foot withdrawal response was considered as a sign of cold allodynia.4446 The Hargreaves test is used to measure thermal hypersensitivity.47 Heat is applied by a high intensity beam to the plantar surface of the hind paw of the animal and pawwithdrawal latency is measured. As compared to mechanical stimuli, heat stimulates other cutaneous receptors. Consequently, specic categories of peripheral axons, including thermo-sensitive and nociceptive bers, can be excited.22 The pain threshold is genetically inuenced by the radiation properties of the skin, namely reectance, transmittance and absorbance and the skin conduction properties. Chemical Tests The Formalin Test. The formalin test explores an inammatory pain condition by subcutaneous injection of diluted formaldehyde into the plantar surface of the hind paw that causes a characteristic pattern of behavioral responses such as lifting, and licking or biting of the injected paw. In this test a rst period of increased response lasting from 5 to 10 minutes, dened as the early phase, is followed by a decreased response for another 5 to 10 minutes. Between 20 to 50 minutes post injection, a second response phase can be delineated.

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This second phase is the reection of a tonic pain stimulus challenging the more central spinal processing neuronal structures.48,49 Other Chemical Tests. Chemical stimulation involving the administration of algogenic agents such as carageenan and zymozan, represents a slower form of stimulation compared to formalin. These respond progressively with a longer duration, and have mostly an inescapable character once they have been applied with painful nerve damage models.22 Animals tend to change their response with stimulus repetition, anxiety (in novel environments) and with a circadian clock.50,51 The accuracy of these tests increases with the simplicity of the behavioral analysis during the test condition. All of these behavioral traits have a signicant genetic component in rodents.52 These inuences will be discussed in the following paragraphs.

For further information we refer to the following articles: Mogil 1996,15 Mogil 1998,62 and Wood 2000.63 Sensitivity of Different Species, Strains and Substrains. An important clinical observation of neuropathic pain is its variability seen from individual to individual, even with identical lesions. Most of the animal models of neuropathic pain have been developed in rodents. Studies in animals using recombinant inbred strains, selective breeding, and targeted or spontaneous mutations have shown that some interesting aspects of pain are heritable, for example a high or low sensitivity to opiates,6466 high and low analgesia in the swim stress tests,67,68 or a high or low autotomy behavior in some pain models.69 Inbred strains of mice and rats, which show only a variability in genotype, show important across-strain differences on biomolecular assays of basal nociception, in models of inammation and neuropathic pain.52,69,70 Animal models for other conditions with associated pain problems, such as rheumatoid arthritis, show genetic based traits for severity and chronicity.71 Pain behavior can be measured in all species, it needs to be stressed however that major differences in receptor structure exists between species.72 For example, the NK1 receptor of gerbils resembles most the human receptor.73 Different defects in genes for tissue signals are described including defects in IL-674 or in products of the pro-opiomelanocortin gene including b-endorphin.75 This knowledge is important in selecting a model for the evaluation of molecules with an activity on specic receptors. Genetic differences between different rat strains were described by Sommer.76 Within the same species large variations in response to induced neuropathic pain have been noted between different strains. Yoon et al.8 compared 7 different rat strains for their reaction to neuropathic pain. Different strains of rats showed different degrees of behavioral signs of neuropathic pain. A similar difference was also found between SpragueDawley rats from two different sources.8 Lovell et al.77 studied differences in neuropathic pain behavior between strains and found that outbred animals showed more neuropathic pain behavior than inbreds. This group suggests that strain-related alterations in proinammatory cytokine release from macrophages near the site of nerve injury affect the magnitude and duration of neuropathic hyperalgesia. Different strains of mice can show different degrees of Wallerian degeneration, in consequence a different onset of thermal hyperalgesia can be observed.76 Species can

FACTORS INFLUENCING NEUROPATHIC PAIN MODELS

Predictivenessin terms of clinical applicabilityis an absolute requirement in nociceptive tests for three reasons: (1) to understand the basic mechanisms underlying pain and analgesia and (2) to search for new molecules with therapeutic value and to avoid false positives and false negatives,39 and (3) to evaluate the right molecule in the right clinical condition. Several factors may inuence pain behavior. Pain sensitivity can be inuenced by inherited genetic variation.15,53,54 Other organismic variables affecting nociceptive sensitivity are ontogeny,55 aging,56 weight,57,58 and hormonal status.59 Broadly, the inuencing factors can be divided in genetic factors, which are also designated as internal factors and environmental or external factors. InternalGenetic Factors The wide variation in clinical pain sensitivities and analgesia together with the evidence for gender differences in pain have shown that there may be denite heritable traits for pain.15,60 The progress in molecular technologies makes it likely that genetic traits for pain and analgesia may be detected in the coming years.61 If an individuals pain related genes could be analyzed and dened a specic pharmacological treatment could be developed.61 The specic technical aspects of genetic evaluation methods or techniques are not described in this article.

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present specic behaviors. Specic selective breeding of a specic behavior can induce traits which can, after several generations of selective breeding, be interpreted as a genetic factor.69 Studies show signicant correlation between observed pain and behavior in this same families.78,79 These animals exposed to a variety of environmental stressors displayed a lower sensitivity to pain compared to non-related animals, and it has been hypothesized that endogenous pain inhibitory circuitry in the central nervous system (CNS) exits to mediate this phenomenon.15 Webster mice can be bred for high and low analgesia, a trait that is supposed to be highly heritable because mediated to a large extend by relatively few gene loci.15 There was no indication of a sex linkage. A recessive inheritance of the autotomy trait was observed, indicating that autotomy is a Mendelian trait, transmitted primarily by a single autosomal recessive gene with minor modiers.69 Considerable evidence suggests that different types of nociception are mediated by separable physiological mechanisms. The genetic mediation of sensitivity to these pain modalities is shown to be different. Genetic factors underlying the sensitivity to opiate inhibition of different pain modalities are divergent. Circadian Rhythms. There exist circadian rhythms of nociception which are strain dependent.8082 Important inuences of genetic determined variations can be detected, such as daytime restriction of food availability phase-shifts the diurnal periodic plasma corticosterone concentration in rats.83 Variations in the pulsatile characteristics of hypothalamo-pituitary-adrenal reactivities in relation to differential stress responses are described between different rat strains.84,85 Genetic Manipulated Animals. Intense research is ongoing to discover effective treatments based on procedures to either selectively remove key neurons in the dorsal horn that drive neuropathic pain, delete appropriate genes, or design drugs or gene therapies with selective neurotoxic effects86,87. The candidates for testing usually come from knowledge of pain physiology and pharmacology, including opioid, peptide or NMDA receptors. The hope is that unimagined aspects of pain physiology may be discovered in this way. Antisense technologies can demonstrate that specic receptor activation plays a role in neuropathic pain states. Of course in these specic techniques the observation of side effect liability is of utmost importance.88

Because the total genome of the mouse is know and these are small and easy to manipulate animals most of the genetic studies are performed in this species. In mice all the receptive behavioral traits have a signicant heritable component comparing across traits, each animal strain has its own characteristic pattern of responsiveness.52 Early genetic studies revealed a surprising degree of genetic variation among common mouse stocks.89 The knowledge of the basal nociceptive sensitivity of commonly used inbred animals is in most cases lacking.15 Data comparing inbred strains have considerable utility as a tool for understanding the genetics of nociception and a particular relevance to transgenic studies.52 Mice have the advantage of being small sized, which renders them amenable to large genetic experiments. Moreover there already exists a large number of inbred mouse strains with carefully catalogued pedigrees.90 The genetic linkage map for this species is more dense than for any other non-human mammal.91 In recent years, the advances in molecular biological technology have induced studies at the level of genes. Exogeneous genes can be added to the genoma or endogeneous genes can be inactivated, both leading to transgenic animals. By denition in those animals where genes were added the term knock-in is used and those animals were specic genes are inactivated the term knock-out is used. Transgenic knock-out technology is practical in mice; behavioral variation can be the basis of genetic studies. Elimination of the protein kinase Cg gene from mice eliminates the neuropathic pain response to partial ligation of the sciatic nerve but does not affect their acute reexes to noxious thermal or mechanical stimuli.92 The mapping of human genetic traits, in combination with animal genetic studies and the new technologies for nucleic acid micro arrays should rapidly give a better diagnosis and treatment for pain.61,93 Several knock-out models have been bred with the objective to study a specic genetic controlled characteristic. The use of knock-out mice induces, however, an automatic compensation mechanism that aims at reinstalling the homeostasis of the model. This means that the basic physiological mechanism does not necessarily resemble the wild type animals reaction, which can strongly inuence the interpretation and conclusions of the test.62 Specic models of knock-out neuropathic pain show that behavioral changes of neuropathic pain are strongly inuenced by the specic knock-down of certain receptors such as the P2X3 receptor.88 Seven days after spinal nerve ligation, the P2X3 receptor

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expression decreased dramatically in small diameter neurons of the L5L6 DRG ipsilateral to the injury, whereas P2X3 expression in large diameter neurons in L5L6 DRG as well as in both small and large diameter neurons in L4 DRG was not altered94. In the future, inhibitor RNA (iRNA) techniques will reduce the bias caused by the compensatory genetic mechanisms in knock-out animals. Transgenic Studies. The advantage of the transgenic approach over conventional pharmacological and antibody-based tools in the investigation of the function of proteins is the complete selectivity obtained in a knockout animals. Many protein receptors, especially those of the same gene family, exhibit overlapping properties such that truly selective ligands are unavailable. However, the nucleotide sequence similarity of related genes is in no way an obstacle to virtually foolproof gene targeting. The main disadvantage of this transgenic approach is that interpretation of phenotypic ndings from KO mice is complicated. One obvious problem is that gene disruptions may be little, although this outcome is surprisingly rare. Mammalian genomes contain less then 40,000 genes. This nite number of expressed proteins, and their interactions with the environment yield all the complexity of homo sapiens. A clear implication is that genes have multiple functions (pleiotropy) and that they and/or their protein products interact to produce biological phenomena (epistasis). The rarity of lethal gene disruption suggests that considerable genetic redundancy (compensation) exists as well. All these have important implications for the interpretation of KO studies.62 The genetic study of analgesics and the heritability of antinociception is an important part of pharmacogenetics. Each individual drug activates a unique receptor, but suggestions are made that the potency of each drug can be affected by a common set of genes. These genes may affect antinociception indirectly via a primary action on baseline nociceptive sensitivity. In the case of acetaminophen there are studies suggesting the existence of gene factors affecting pharmacodynamics rather than pharmacokinetics.14,95,96 Interactions of mouse genotype with the specic laboratory environment in which traits are examined may have hampered the replicability of transgenic studies. In one of these studies heroic attempts to standardize all facets of the laboratory environment in three different laboratories including acquisition, husbandry and

testing protocols were made; nevertheless, signicantly different responses on various behavioral tasks were obtained at each site.97 Polymorphism and Pain Sensitivity: Is There a Specic Predisposition to Pain? Since a high natural genome sequence variability and complex genotype-phenotype relationships are described specic receptor gene variations will result in different clinical sensitivities for a certain effect.98 For example, the mu opioid receptor (mOR) gene variation is demonstrated to result in a variation in the sensitivity for substance dependence in human subjects99 and strain comparison studies in mice identied the simultaneous existence of reduced antinociceptive responses to morphine and lower levels of mOR expression in some mouse strains.100 The mORs play an important role in normal nociception and normal opiate drug responses. Changes in these mOR densities can produce differences in both nociceptive responses and in the modulation by opiates. In addition, allelic variants at the mOR locus can contribute to variations in the effects of opiates in animals and human subjects. Specic information about mOR gene polymorphisms can predict the efcacy of individualized drug treatments100 and growing evidence indicate that interindividual differences in the experience of pain and analgesic responses can be caused by these allelic variations. Thus, genomic variations can explain the analgesic variability to gender differences in animals and human subjects101 and to several drugs.54,102 Gender. Female Sprague-Dawley (SD) rats demonstrated signicantly greater mechanical allodynia as compared to male SD rats.103 There were no statistically signicant differences overall between strains or when the same gender was compared between strains. This is an important observation in order to enable comparisons of behavioral studies of nociception between laboratories using SD or Holtzman rats of the same sex.103 Compared to males, there is a modestly increased sensitivity to noxious stimuli in females belonging to a number of species, including rodents and humans.60,104,105 In humans the analgesic sensitivity for a specic opioid can be gender specic: kappa opioids are more potent analgesics in women compared to men.106 Specic spinal kappa opioid receptor dependent mechanisms leading to analgesia were only found in female rats. Sex steroids seems to play a major role in the physiology of this mechanism.107

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Age. The prevalence of some neuropathic pain disorders declines with age.108 This nding has been conrmed by Crisp et al.109 who compared the effect of age on thermal hyperalgesia and tactile-evoked allodynia in the CCI and PSNL model of peripheral mononeuropathy in the rat. Aged rats displayed a more vigorous allodynic response post PSNL than did younger counterparts. In addition, older animals failed to develop signicant tactile-evoked allodynic responses post CCI.109 One explanation for this phenomenon is that the brous sheath surrounding the sciatic nerve is thicker in older rats, conceivably obstructing the development of CCIinduced allodynia by reducing the extend of myelinated and unmyelinated ber loss.110 Recent investigations using CCI and PSNL models to assess age-related changes in spinal microglia activation discovered that OX-42-ir, a marker for microglia activation, was elevated in the dorsal horn of the rat spinal cord post CCI more so than post PSNL. The injury induced increases of the marker, were attenuated in aged rats compared to young animals, supporting the hypothesis that the reactive immune response to neuropathic pain, decreases with age.109,111 ExternalEnvironmental Factors Clinical management of chronic pain is now accepted to require a multidisciplinary approach, allowing evaluating the contribution of somatic, psychological and social factors. Likewise, in animal models for neuropathic pain, environmental factors have been demonstrated to interfere with the study results. In human beings, the genomic inuence on behavior is currently unknown as the reactivities to environmental factors could be genomically predisposed. In humans, only a very low heritability of 10% was found in the determination of non-pathological experimental pain or specically in monozygotic twins for pressure pain thresholds.112 This nding reinforces the important role of environmental inuences including family dynamics, learning and adaptation of pain behavior.79,113 It is still unclear in most species whether genetic or environmental factors are the more important in certain experimental pain conditions.52 Even during development environmental factors may inuence pain behavior. This is described in prenatal stress to the mother,114,115 prenatal lead exposure,116,117 and even neonatal handling.118120 Environmental factors inuencing the pain behavior in adulthood, include stress exposure,121 housing density, diet,122,123 the presence of magnetic elds,124 and daily

activity rhythms.125 Specic test conditions inuencing pain behavior are its timing during the day and novelty stress associated with the testing room.126 Important gender stress differences can be expected. For female rats, isolated housing is a stressor and for male rats, crowding is a stressor.127 Further, aggression of group housed males128 and social defeat associated with that aggression is a major stressor that can affect nociception and analgesic sensitivity.129 Timing of the Experiment Post Induction of Neuropathy. Currently the 3 most frequently used neuropathic pain models are the chronic constriction injury model (CCI)2 the partial sciatic nerve ligation model (PSNL)5 and the spinal nerves ligation model (SNL).12,130 The intensity and duration of these elicited behaviors is different from one model to another as demonstrated in a study comparing the 3 models.12 The rst day after surgery animals in the SNL and the PSNL group show a high response to mechanical stimulation (Von-Frey test). CCI intervention results also in mechanical allodynia, the intensity of the response increases gradually to peak approximately at the same time as after the other interventions (2 weeks). Thermal allodynia, on warm and cold plate, is noted the rst day after the intervention in the CCI animals, whereas the duration of paw lifting was only slightly increased in the rst week after the intervention in the two other groups. This effect peaked equally at 2 weeks after the intervention for PNSL and SNL animals. The pain behavior starts decreasing in the CCI animals form the third day after the intervention. In the CCI model, clear signs of neuropathic pain behavior become evident and stable after a mean period of at least 4 to 7 days. This period coincides with the progressive induction of the Wallerian degeneration. In non-neuropathic inammatory pain models, the presentation of pain occurs much earlier.31 Circadian Rhythms. In rodents a decreased sensitivity in the dark phase compared to the light phase is reported for thermal nociception.131 Time regulated feeding restriction regimen did not alter the circadian variations of corticosterone in rats.132 Whether differences can be observed within a photo phase is until now not clear. Circadian and chronobiological inuences on specic pain behaviors or hormonal parameters are important, though not yet fully unraveled.133

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Inuence of Season and Humidity. Another potential biological difference between strains of rats is that different strains of rats may show different behavioral sensitivities in different seasons of the year.134 Sensitivity to a noxious thermal stimulus is higher in conditions of high humidity in natural circumstances in temperature climates would correspond to late spring, summer and early fall. Circannual and weather effects on pain related traits are not entirely known. Circannual variation in concentration of endorphins in human cerebral spinal uid has been reported with lower values in summer and higher values in winter.135 Seasonal inuences have been shown to affect analgesic magnitude and even neurochemistry in rodents.136,137 Seasonal and humidity effects may have measurable inuences on any number of behavioral traits.138 Inuence of Order and Testing. The phenomenon of inter-animal communication and the order of testing can inuence experiments.14 How this communication is achieved is unclear, via chemo sensation or a pheromonal alarm substance139 or ultrasonic vocalization or alarm cry.140 The release of alarm substances and ultrasonic vocalization are known to be genotype dependent in rats.141 The effect on animal behavior of the order of different consecutive sensorial and pain tests is no yet evaluated. Diet. Nutrients consumed by animals peri-operatively and postoperatively affect nociceptive measures at baseline and in neuropathic pain models.123,142145 Observation of a weakened sensory response after the otherwise standardized and validated PNSL model, prompted Shir et al.145 to evaluate the potential inuence of the diet. The addition of soy to the animal food resulted in a weaker pain response in the test animals. When soy was omitted and casein was added to the diet the robustness of the model was restored.145 Social Variables. Social factors must be considered in behavior-related research on rodents.146 The social conict analgesia has been described in mice, illustrating an analgesic effect in non-aggressive group-housed animals when intruded in a cage of a singly-housed resident, which shows a modest hyperalgesia.147,148 Maternal behavior and stress reactivity can be transmitted non genomically across generations.149 In rats the psychosocial-behavioral variable, the identity of the cage mates, can largely over-ride genetic pre-

disposition to pain behavior. This was demonstrated using selection-line rats that consistently show high (HA) or low (LA) phenotype (autotomy) in the neuroma model of neuropathic pain. Normally, HA animals show autotomy after nerve injury while LA animals do not. However, when caged together with HA rats, LA rats showed high levels of autotomy. This occurred even when the individual HA cage mates were familiar preoperatively, and it did not depend on the actual performance of autotomy by the HA rats. Cage bedding soiled by HA rats was sufcient to induce a modest level of autotomy in LA animals.69 Housing and Manipulation. Although an animal room might conform to the temperature and humidity guidelines, animals may suffer from heat stress if there is overcrowding and an inappropriate cage design.150 Exposure to bright light, often resulting from cages being high on a rack, can induce retinal degeneration and decreased growth rates in various strains of rats and mice.151 When rabbits are handled in a consistent and friendly way as opposed to the more usual laboratory procedure of simply picking them up and restraining them, arteriosclerosis is reduced.152 When animals are moved to a novel cage, increases in blood pressure, heart rate and locomotor behavior occur, which are indicative of a stress response.153 Noise and vibration of building constructions, have caused major problems with rat behavioral studies and experiments requiring control unstressed rats.154 Noise from personnel activity in the animal facility leads to increased micro vascular permeability and disruption of the intestinal lining in rats.155,156 These factors may alter the animals stress level and its behavior. The effects of housing density and photoperiod may be strain dependent.80 Inuence of Cage Density. Housing in the laboratory setting is known to affect any number of morphological and biochemical variables,157 notably including plasma corticosterone levels.158 Adult rodents show a hypo algesic effect of isolation on thermal nociception and analgesia.159,160 Inuence of Sexual Activity. Sexual activity exerted an important inuence as factor that interacts with the environment. Biological sex differences may manifest themselves in differential sensitivity to environmental factors.14

336 vissers et al.

External Stress Factors. Several reports mention stress induced analgesia in rats.147,161,162 Some of the factors eliciting analgesia in rodents are: brain stimulation, body pinch, centrifugal rotation, classical conditioning, copulation (males), 2-Deoxy-D-glucose, electric shock, forced swimming, heat exposure, hypertonic saline, insulin, irradiation, novelty, predation, presence of natural predator, restraint/immobilization, social conict, social isolation, stress odors, tail pinch/pressure, and vagal stimulation. Although all of these stimulus situations undoubtedly involve varying degrees of physical/psychological stress, the term environmentally induced analgesia is preferred to that of stressinduced analgesia. The most important factors have already been described above. It is important to mention other situations that might inuence the test animals pain behavior in order to improve standardization of the test conditions. Although, again the inuence of the genotype in all these situations must be further studied. Inuence of Experimenter. The high importance of the experimenter in the test conditions was found by Chesler and Crabb.14,97 Neither the experimenters age, sex, or experience level seemed to correlate with the observed differences. The experimenter effect is not of the same magnitude in all strains. It seems likely that differential animal handling induces different levels of stress in the subjects and that this is responsible for the experimenter effect. It may be possible that different experimenters affect latencies on these test by a more generalized mechanisms for example their particular odor.14 Genetic/environmental Correlations and Interactions Differential effects of the environment could result in over or underestimation of genetic effects. Genetic and environmental factors are interacting importantly to inuence thermal nociceptive sensitivity.14 In the study of Crabb97 genotype was highly significant for all behavioral studies accounting for 30% to 80% of the total variability and several historically documented strain differences were also seen in this experiment. In general the authors conclude that very large strain differences are robust and are unlikely to be inuenced in a major way by site-specic interactions. However, a more cautious reading suggests that for behaviors with a smaller genetic effects, like those seen in gene knock-outs, there can be an important inuence of environmental conditions specic to each individual

laboratory and the specic behavioral effects should not be uncritically attributed to genetic manipulations such as targeted gene deletions.97

IMPACT ON STUDY CONDITIONS

Some have argued for standardization of environmental conditions to improve replicability and comparison of results across laboratories.163165 Standardization can lead to ndings with low possibility to generalize and low external validity, essentially rendering experiments uninformative and argues instead for systematic variation of the testing environment.166 Experimental variation introduces a challenge to the interpretation of ndings obtained from different studies, since both analgesia and hyperalgesia are by denition changes from the baseline. In light of ceiling effects, the cut-off imposed for ethical reasons and oor effects, the minimal time taken for a response to be performed and recorded, the baseline values themselves impact greatly on estimates of analgesic and hyperalgesic potency and efcacy.14 This problem is magnied in those studies comparing more than one subject population. A complete knowledge of factors inuencing baseline measurements becomes important.14 The tail withdrawal latency is higher when mice are restrained in plexiglas chambers for the entire experiment, even after habituation, compared to when they are removed from their home cage immediately prior to testing and restraint acutely.167 Even when all parameters are controlled, variability remains. This interindividual variation is due to organismic and environmental factors and their interaction.14

CONCLUSION
Overall, the following recommendations concerning the genetic and environmental inuences can be considered: if possible, genotypes should be tested in different laboratories and evaluated with different test of a single behavioral domain before concluding that a targeted gene inuences a specic behavior. This implies that genetically predisposed phenotypes can be affected by laboratory specic effects. Until now, it is unclear whether standardization of behavioral essays in those laboratories would markedly improve future replicability of results across laboratories.164,165 A strict standardization and description of the pool of variables studied in controlled experiments is necessary to differentiate between heritability and environmental inuences.168 The variation in nociceptive sensitivity is caused by environmental factors and their

Internal and External Factors Affecting the Development of Neuropathic Pain in Rodents 337

interaction with genes. Once the relevant genes are located, clinical important information that may facilitate individualized pharmacologically and behaviorally based treatment strategies can be explored. Even when genetic factors would only play a minor role in the pain sensitivity of humans, genetic studies on pain are important. These studies will demonstrate which specic components in the biology of pain are sensitive to specic changes. This information can be important to develop novel analgesic strategies. Finally, the knowledge of these phenomena can help in the unraveling and the treatment of chronic neuropathic pain, and hopefully in development of specic algorithms to prevent chronic neuropathic pain.

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