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

Critical Steps

Choice of the animal model for nociception measurements

Variability in nociceptive and analgesic sensitivity among mice strains has been examined (reviews ^14-16) reviews using various pain models differing in their etiology (nociceptive, inflammatory, neuropathic), modality (thermal, chemical, mechanical), duration (acute, tonic, chronic) and site of administration (cutaneous, subcutaneous, visceral). When compared to other strains, C57BL/6J (“J” for Jackson Laboratory) mice became a popular animal model for pain studies as they exhibit a high basal nociceptive sensitivity^17,18 and a moderate analgesic response to opiates^14,19. Following chronic morphine treatment, they also develop significant analgesic tolerance^20,21, hyperalgesia^21,22 and dependence^20,23.

Here, experiments were performed on C57BL/6N Tac mice (“N” for National Institute of Health and “Tac’ for Taconics farm) which belong to a separate branch of the B6 lineage. Although C57BL/6 mice have been long considered as interchangeable, recent studies pointed to significant behavioral differences among C57BL/6J and C57BL/6N strains²⁴. In particular, the lower sensitivity of the three C57BL/6N substrains (including the Tac one) to acute thermal pain may be regarded as an advantage for testing this phenotype.

Male mice were selected as the vast majority of pain studies, using mice as the animal model, are performed on juvenile males²⁵. In our hands, they provided robust and reproducible data when examined from the analgesia or hyperalgesia points of view. Occasionally, we noticed a tendency for C57BL/6N females to provide more variable responses, both in the TIT and TPT tests. Although this observation may reflect natural variations linked to the hormonal status of females, overall mechanisms underlying sex differences in pain and analgesia still remain a matter of controversy. Some aspects of this hot debate will be briefly presented in the next ‘Limitations of the technique’ section.

Animal habituation

Mice were first allowed to get accustomed to the animal facility during one week. Similar to any other behavioral study, testing was performed following a 3 day-acclimatization period (Figure 1, step A). As nociceptive tests are sensitive to stress, first measures may give longer latencies than subsequent ones, especially in non-habituated mice^26,27. The habituation step allows also the obtention of more stable nociceptive response values within the same day and between days Figures 2 and 4. To reduce circadian effects on nociceptive and analgesic sensitivity^28,29, all testings were conducted between 10:00 am and 4:00 pm.

Selection of nociceptive tests

Nociceptive tests use either thermal, mechanical, chemical or electrical stimuli (review ^26,27,30. Their choice is critical as different nociceptive modalities may be processed through different nociceptors and fibers^18,31,32.

We selected the tail immersion test (TIT)³³, a modified version of the classical tail flick test developed by D’amour and Smith³⁴, and the tail pressure test (TPT), adapted from Randall and Selitto³⁵, as examples of thermal and mechanical modalities to study morphine-induced analgesia, hyperalgesia and tolerance in mice. Both tests have been widely used in rats. A cut off time was systematically defined to avoid or limit the risk of tissue damage.

Morphine-induced analgesia, hyperalgesia and tolerance

Morphine, the prototypical mu-opiate agonist, was selected here as it is a potent analgesic and OIH-inducer, both in humans and mice^1,2,36. Morphine analgesic potency is known to vary with mice strains, routes of administration and nociceptive modalities. In C57BL/6 mice, reliable analgesia is usually obtained following subcutaneous injections of morphine in the 1-20 mg/kg dose range^14,21. Accordingly, we chose to study acute analgesia following a single administration (s.c.) of morphine at 5 mg/kg, close to its ED₅₀ value (7-20 mg/kg) assessed from thermal nociception^19,21.

Repeated morphine administration is often accompanied with analgesic tolerance (evidenced either from a rightward shift of the dose-response curve or from a decrease in analgesic response amplitude or duration) and hyperalgesia (an exacerbated sensitivity to painful stimuli evidenced from a decrease in basal nociceptive value). Both adverse phenomena depend on rodent strains, on the nature of the opiate compound which is selected and its dosage, on treatment duration and on nociceptive modalities²¹. For example, experimental paradigms to study tolerance and hyperalgesia consist in daily administration of high and constant (20 to 40 mg/kg per day)²² or of escalating (up to 50 or even 200 mg/kg)^20,21 morphine doses. Accordingly, we promoted the development of hyperalgesia and tolerance in C57BL/6 mice through daily morphine administration (5 mg/kg; s.c.) over a 8-day period. This moderate morphine dose was preferred over higher ones to better mimic clinic usage.

Set up of TIT operational window

A possible pitfall in TIT might be related to the role of the tail in the thermoregulation of rodents^26,37. As ambient temperature is a key factor in nociceptive response variations, it should be kept constant (here at 21 °C) throughout experiments³⁸. Heat intensity is usually set up to detect a nociceptive response within 5 to 10 sec²⁷. Indeed, greater latencies may increase the risk for monitoring animal movements unrelated to the nociceptive stimulus, whereas shorter ones may reduce the differential power of the test. We performed TIT measurements at a fixed temperature of 48 °C. Tail withdrawal latencies were close to 9 sec (basal nociceptive value) and varied from 4 sec (hyperalgesia) to 25 sec (maximal analgesia; cut off). In addition to practical reasons, measurements of nociceptive response values at a fixed temperature may a priori involve the same repertoire of nociceptors and circuits, thereby facilitating data interpretation.

Possible Modifications

Optimization of the TIT operational window for analgesia and OIH measurements

When focusing on an analgesic response, low baseline values (higher heat intensity) may favor the detection of a delay in the response. In turn, to address the consequence of a painful stimulus or the development of OIH, higher baseline values (lower heat intensity; here 48 °C) may facilitate the detection of faster responses Figure 4.

Although we found morphine at 5 mg/kg a convenient dose to induce a robust analgesic response Figure 3 and to promote (upon repeated administration) significant hyperalgesia Figure 4, its dosage may be adapted as mentioned before (Critical step : Morphine-induced analgesia, hyperalgesia and tolerance). For example, lower doses may be used to reduce analgesia amplitude (thereby avoiding cut-off limitations) whereas higher doses may be chosen to accelerate hyperalgesia onset and increase its amplitude.

Overall, optimization of the ‘nociceptive window’ should be adapted to the genetic background of mice under study and take into account the possibility for the involvement of distinct arrays of nociceptors and circuits.

Alternative opiate agonists (fentanyl, remifentanyl)

Although most clinically used opiates target the mu-opioid receptor as agonists, they differ considerably with respect to their pharmacological properties both in vitro and in vivo. For example, remifentanyl and fentanyl, in marked contrast with morphine, behave as full agonists and promote internalization of mu-opioid receptors³⁹. Opiate analgesics such as morphine and fentanyl have half-lives in the range of hours⁴⁰, while remifentanyl has an ultra-short half life of several minutes⁴¹. In humans, best evidence for OIH is from patients who received opiates during surgery, including short-acting compounds such as remifentanyl^2,42. Thus, fentanyl and remifentanyl may be valuable tools too to study the development of hyperalgesia and tolerance in mice, under TIT and TPT paradigms.

Alternative modes of induction of OIH (chronic vs acute administration)

OIH is seen in humans and animal models as a consequence of opiate administration, whether at very low or extremely high dosages^1,2. We report here on OIH development following chronic treatment of mice with moderate doses of morphine. Several days of treatment of C57BL/6N mice were necessary to evidence a clear and reproducible hyperalgesic state Figure 4. Daily morphine injections could be adequately replaced with implanted morphine pellets : upon their removal, both thermal hyperalgesia and mechanical allodynia have been already reported in mice⁴³. Infusion of opiates through a micro-osmotic pump is another possibility⁴⁴. In rodents, long-lasting hyperalgesia is also achievable following acute administration of fentanyl using a protocol mimicking the use of this mu-opioid agonist in human surgery^36,45,46.

Limitations of the Technique

Animal species and models for pain

Comparative studies of numerous mouse strains provided evidence for great variations in nociceptive responses to painful stimuli^17,31,47 and in OIH levels following 4-days morphine treatment²². Whether mechanisms underlying pain processing and modulation in animal models (mice and rats) are relevant for chronic pain patients remains a fundamental and opened question. Thus, much caution should be paid to the interpretation of animal data and to their predictive validity for humans¹⁶.

Sex differences in pain and analgesia

Most preclinical studies on animal models for pain have been conducted on male rodents^16,25,48. Despite this selection bias, the emerging view was to consider males as better responders to opiate analgesics^49,50, less prone to develop opioid-induced hyperalgesia ^51,52 and more tolerant to morphine analgesia⁵³ than their female counterparts (review ⁵⁴). However, sex differences with regard to nociception and analgesic drugs efficacy do not resume in such ‘a one size fits all’ paradigm. Indeed, a wealth of data now indicates that numerous variables may influence the magnitude and direction of sex differences such as opioid drug efficacy and selectivity, nociceptive assay, genetic background, age, gonado-hormonal status or social interaction^48,54. In humans, clinical pain is more prevalent in women but whether this fact reflects actual sex differences remains a matter of debate^48,55,56. For example, global analysis of fifty clinical trials indicated no significant differences in analgesic properties between genders whereas meta-analyses performed on patients-controlled subjects pointed to a significantly greater opioid efficacy in women⁵⁷.The latter observation, which markedly contrasts with what has been found in rodents, again raises several questions regarding the origin for such divergences^16,48,55,57. Altogether, sex differences in analgesia do exist and merit further focus on underlying mechanisms and clinical implications.

About nociceptive tests

The tail withdrawal test is a spinal reflex but it may be subject to supraspinal influences⁵⁸. TIT is relatively easy to perform on rats but requires more expertise in mice. A potential difficulty is to maintain the mouse in a correct posture without inducing unwanted stress. The proposed protocol may be adjusted according to cohort size. 16 animals (8 control and 8 treated) are easily managed as far as measurement of their basal nociceptive response values (using TIT first, then TPT for the whole series of mice) is under concern. Monitoring analgesia time courses requires the establishment of a precise time schedule and the evaluation of the maximal number of animals that may be tested (TIT first, then TPT) within the imparted time interval (here 30 min). The whole cohort of animals may thus be divided into subgroups to allow the experimenter to respect kinetic limitations.

Significance of the Technique with respect to Existing / Alternative Methods

OIH in rats versus mouse

Rats have been extensively used to study opioid analgesia, hyperalgesia and tolerance, following acute or chronic opiate administration^46,59-61. Indeed, for several practical reasons, they may be considered superior to mice as an animal model for pain experiments^16,61. However, until recently, the generation of genetically modified rats was not a straightforward procedure. As numerous genetically modified mouse strains are already available, our model offers the opportunity to study the contribution of numerous individual genes in OIH and tolerance development in mice.

TIT and TPT versus other nociceptive tests

TIT is a variant of the tail flick test, the most obvious difference being the area of stimulation. In contrast with radiant heat, immersion of the tail into hot water leads to a quick and uniform increase in its temperature. Compared to other forms of thermal nociception testing (hot-plate or Hargreaves tests), TIT provides fairly reproducible results both across and within subjects.

TPT is a very popular test for the study of mechanical nociception^26,27,35 which probably involves distinct nociceptive fibers and molecular transducers then TIT³². It provides quick and reliable measurements⁵⁹ but requires some expertise from the experimenter and large animal cohorts. As an alternative to the analgesimeter used in the present study, other procedures or apparatus relying on strain gauges do exist (review ²⁷). TPT is best suited for studying mechanical hyperalgesia whereas von Frey filaments are usually taken to evaluate mechanical allodynia (review ²⁷).

Future Applications or Directions after Mastering this Technique

The experimental OIH/tolerance model we present here may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of pain. It also provides a model system to investigate the effectiveness of potential therapeutic agents to relieve chronic pain.