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Evaluation of anxiety-like behaviour in a rat model of acute postoperative pain

Kouya, Francois*; Iqbal, Zohaib*; Charen, Daniel; Shah, Mansi; Banik, Ratan K.

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European Journal of Anaesthesiology (EJA): April 2015 - Volume 32 - Issue 4 - p 242-247
doi: 10.1097/EJA.0000000000000052
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An estimated 46 million inpatient surgical procedures are performed each year in the United States alone.1 At least half of these patients suffer from unrelieved pain,2 which can often lead to comorbidities, such as anxiety, depression, restlessness and sleep deprivation.3,4 It has been shown that postoperative anxiety is associated with longer hospital stays and increased treatment costs.5

In preclinical studies, rodent models have been useful in identifying mechanisms and therapeutic targets for postoperative pain.6 In contrast with clinical studies that evaluate both the subjective and affective components of acute pain,7 most rodent studies assess only the pain-like behaviour following acute heat or mechanical stimulation, or guarding behaviour. However, several behavioural paradigms have been used in recent studies to assess anxiety-like behaviour in chronic pain models.8 These paradigms are based on an animal's innate aversion to stressful situations such as bright illumination. Using these tests, previous studies have shown that anxiety-like behaviour is present in rat models of pain associated with varicella zoster virus,9,10 HIV-induced neuropathy,10,11 persistent inflammatory pain by intraplantar injection of complete Freund's adjuvant,12 nerve injury caused by partial sciatic nerve ligation13 and after spinal nerve ligation.14 In contrast, Kontinen et al.15 and Hasnie et al.16 did not observe anxiety behaviour in the rat and mice models of spinal nerve ligation.

We set out this study to evaluate unrelieved acute pain-associated anxiety-like behaviour and the effects of low-dose morphine on this behaviour. The two most widely used tests for anxiety assessment, the elevated plus maze (EPM) and light–dark tests were applied to assess anxiety levels in the rat model of acute postoperative pain.

Materials and methods


A total of 42 male Sprague–Dawley rats (Harlan, Somerville, New Jersey, USA), each weighing 280 to 300 g at the start of experimentation, were divided into eight groups. Rats were housed two to three per cage in a room with a 12/12 h light/dark cycle. Food and water were available ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Seton Hall University, and were in accordance with the Ethical Guidelines for Investigation of Experimental Pain in Conscious Animals, issued by the International Association for the Study of Pain.17

Plantar incision model

The plantar incision model was prepared according to the method described by Brennan et al.18 Anaesthesia was induced by placing each animal within a plexiglass box containing 5% isoflurane and air. After loss of righting reflex, anaesthesia was maintained by administering a 2 to 3% isoflurane and air mixture through a tightly fitting nose cone. A 1-cm longitudinal incision was made through the skin, fascia and muscle of the plantar aspect of the right hind paw of anaesthetised animals. The plantaris muscle was elevated and incised longitudinally with the origin and insertion of the muscle remaining intact. The skin was closed with two mattress 5-0 silk sutures. The animals were then housed individually on soft bedding, and allowed to recover. Sutures were removed on the 2nd postoperative day.

Escape-avoidance behaviour in the light/dark test

Anxiety behaviour was tested using a light/dark paradigm, as previously described.19,20 The illumination level was measured by a digital luxmeter (Model# LX 1010B; Mastech, Hong Kong, China). In accordance with the light/dark test, the experiment was conducted in a dark room (0 Lux). A light/dark box with two equal compartments, each 43 × 30 × 30 cm, was placed on an elevated metal mesh floor. One compartment was relatively dark (100 to 200 Lux) with black walls and a black floor, connected through an open door to an intensely lit (6000 Lux) compartment with white walls and a white floor. At the beginning of each 5-min observation session, an animal was placed in the light compartment. Each animal's entries and exits into and out of the light compartment were counted when all four of its paws crossed the plane of the open door.

Escape-avoidance behaviour in the elevated plus maze

The EPM, consisting of two open arms 30 × 10 cm each, two closed arms 30 × 10 × 30 cm each and a centre 10 × 10 cm square, was performed with all arms at an elevation of 50 cm from the floor. Rats were gently placed on the centre square facing one of the open arms, below an illuminated light bulb (15 watts) that was 25 cm above the centre square. The time each animal spent in the open arms was manually measured with a stopwatch during each 5-min testing period.

Assessment of the mechanical hypersensitivity-like behaviour

The rats were placed on metal grid floors (IITC Life Sciences, Woodland Hills, California, USA), which were covered with Plexiglas compartments (14 × 20 × 27 cm). Testing commenced once the animals became largely inactive, demonstrating only occasional grooming behaviour. The animal paws were stimulated with an electronic von Frey aesthesiometer. The device, consisting of a handheld force transducer attached to 0.8-mm polypropylene tips (IITC Life Sciences), was used to apply a stepwise increasing force to the mid-plantar surface of the rats’ hind paws, adjacent to the surgical injury, if present. Stimulation was applied until a paw withdrawal response was observed. The force was recorded from the aesthesiometer's electronic display. The results were expressed as the paw withdrawal threshold (PWT) for mechanical nociception, which was calculated by averaging three to five measurements taken at 2-min to 5-min intervals.

Open field locomotion test

Rats were allowed to acclimatise to the dark (0 Lux) testing room for 15 min. The open-field consists of a wood floor (90 × 90 × 40 cm), marked into 30 × 30 cm squares, and completely surrounded by a rigid opaque paper wall. Rats were placed in the middle square and allowed to roam around the open field for 10 min. A count was kept of the number of squares a rat entered during this time period. In order to be counted as crossing a line, a rat must have crossed a line with all four paws. A diagonal crossing of a line with all four paws was counted as crossing a line as well.

Experimental protocol

Experiment 1: effects of plantar incision on anxiety-like behaviour

Sixteen animals were used to investigate plantar incision-evoked anxiety-like behaviour. The rats were tested for baseline values 3 days prior to surgery. Prior to behavioural testing, all animals were acclimatised to the testing environment for 10 to 60 min. Both incised and unincised rats were tested for PWTs by von Frey hair stimulation, and for avoidance behaviour by the light–dark and EPM tests at 1, 3, 8 and 14 days after surgery or sham operation (only anaesthesia). The time, animals spent in the light box or open arms, was compared between groups.

Experiment 2: effects of morphine (1 mg kg−1) on plantar incision-induced mechanical hyperalgesia and anxiety behaviour

Eighteen rats were randomly tested in three groups. In each group (n = 6), all animals were incised and tested at 1, 2 and 3 days after plantar incision. In each testing day, an animal randomly received either an intraperitoneal injection of saline or morphine 1 mg kg−1. Thirty minutes following drug administration, rats were tested for PWT by von Frey hair stimulation and for avoidance behaviour by the light–dark and EPM test. The data from these three identical experimental groups are combined for analyses (n = 9 saline or morphine). The animals were tested by an investigator blind to the treatment assignments (drug vs. saline). Testing was completed within 2 h of drug administration. Stock solutions of the morphine were refrigerated, warmed and diluted to their final concentrations with saline (0.9%) prior to injection.

Experiment 3: effects of morphine (1 ml kg−1) on rat locomotor activity after plantar incision

Eight rats were tested for open field locomotor activity for 5 consecutive days to allow habituation with the testing environment. Following these sessions, rats were incised in the plantar hindpaw and tested for locomotor activity at 2 h, day 1, day 2, day 3 and day 4 after surgery. On the 1st and 2nd postoperative days, each rat was initially tested for baseline locomotor activity and returned to its cage. After an hour, rats were injected with morphine or saline on the 1st and 2nd postoperative day, respectively. After injection, rats were returned to their home cages 30 min prior to being placed in the open field chamber.

Data analysis and statistics

The mechano-sensitivity data obtained with the electronic von Frey device constitute continuous data as they represent exact noxious threshold force values. These data are presented as the mean ± standard error. A non-parametric Mann–Whitney test was performed to determine differences between anxiety behaviour in the control and incised animals, or incised animals with saline or drug treatment. The time course of open field locomotion over 2 weeks was compared using a one-way analysis of variance (ANOVA) test followed by a Dunnett's test. The differences in the locomotion after saline or drug treatment were compared using a parametric Student's t-test. P < 0.05 was considered statistically significant.


There was no dropout of animals in the current study.

Before incision, two randomly assigned groups of rats had similar thresholds to punctate mechanical stimulation (27 ± 10 vs. 31 ± 7 g, n = 8, P = 0.4) (Fig. 1a). After incising the plantar hind paw, the threshold force required for paw withdrawal behaviour decreased to 4 ± 4, 8 ± 4 and 16 ± 1 g, respectively, on the 1st, 3rd and 8th day postincision (P = 0.0002, unpaired t-test) (Fig. 1a). By 14 days postincision, all rats had completely recovered. Sham-operated rats displayed no significant differences in withdrawal thresholds between daily measurements. One day after surgery, incised rats spent significantly less time than sham rats in the light compartment (Fig. 1b), that is, they showed more anxiety-like light avoidance behaviour on the 1st and 3rd postoperative day when they experienced significant mechanical hyperalgesia (P = 0.02, Mann–Whitney test). Moreover, on the 3rd postoperative day, incised rats exhibited significantly fewer entries into the bright chamber (P = 0.028, Mann–Whitney test) (Fig. 1c). The time spent in the bright chamber returned to preoperative values by the 8th day following plantar incision.

Fig. 1
Fig. 1:
Plantar incision induces anxiety-like light avoidance behaviours in the light/dark box. (a) Rats developed significant mechanical hypersensitivity behaviours after plantar incision on the 1st, 3rd, and 8th day postincision (P < 0.0003, unpaired t-test). Sham-operated rats displayed no significant differences in withdrawal thresholds between daily measurements. (b) Compared with sham-operated rats, the incised rats spent more time in the bright compartment on the 1st and 3rd postoperative day (P < 0.03, Mann–Whitney test). The time spent in the bright chamber returned to preoperative values by the 8th day following plantar incision. (c) At the 3rd postoperative day, incised rats exhibited significantly fewer entries into the bright chamber (P < 0.05, Mann–Whitney test).

In the EPM test, all incised rats (n = 8) spent significantly less time in the open arms on the 8th postoperative day (P = 0.028, Mann–Whitney test) (Fig. 2a). The incised rats generally displayed less exploratory behaviour in the open arms as shown in the intergroup comparison of the count of crossing the middle square in EPM. The count of middle square crossing was significantly reduced in the incised rats by the 3rd postoperative day (P = 0.038, Mann–Whitney test) (Fig. 2b).

Fig. 2
Fig. 2:
Plantar incision induces fear-like open arm avoidance behaviour in the elevated plus maze (EPM) test. (a) The incised rats spent significantly less time in the open arms on the 8th postoperative day (P < 0.03, Mann–Whitney test). (b) The count of middle square crossing was significantly reduced in the incised rats at 3rd postoperative day (P < 0.05, Mann–Whitney test).

After plantar incision, rats displayed normal locomotion as assessed in the open field test (Fig. 3). There was no significant difference in locomotor activity in the open field prior to surgery up until 4 days postsurgery (P > 0.05, ANOVA followed by Dunnett's test).

Fig. 3
Fig. 3:
Plantar incision has no significant effect of rat's locomotor activity in an open field test. (a) There was no significant difference in locomotor activity in the open field prior to surgery up until 4 days postsurgery [P > 0.05, analysis of variance (ANOVA) followed by Dunnett's test]. (b) Effect of morphine (1 mg kg−1 intraperitoneally) on open field locomotor activity at 1 and 2 days after plantar incision.

The pretreatment of intraperitoneal morphine (n = 9; 1 mg kg−1) significantly increased the withdrawal threshold compared with saline at 3 days (P = 0.038) and 8 days (P = 0.014) after incision, but had no effect at 1 day postincision (Fig. 4a). Morphine had no discernible effect on light or open arm avoidance behaviour (Fig. 4b,c). The number of entries into the light compartment was also similar between groups. In addition, after morphine administration, exploratory behaviour, that is, middle square crossing in the EPM was similar at 1, 3 and 8 days after incision. To see whether morphine-induced altered locomotion contributed to above results, the effects of morphine were observed in a group of rats 1 day after incision. Morphine 1 mg kg−1 had no significant effect on locomotion compared with baseline (Fig. 3b).

Fig. 4
Fig. 4:
Effects of morphine (1 mg kg−1) on plantar incision-induced mechanical hyperalgesia and anxiety behaviours. (a) The pretreatment of morphine significantly increased the withdrawal threshold to von Frey hair stimulation compared with saline at 3 (P = 0.038) and 8 (P = 0.014) days after incision, but had no effect at 1 day postincision. Morphine had no discernible effect on light (b) or open arm avoidance behaviours (c).


The current study demonstrates that rats after plantar incision develop an increase in light avoidance behaviour and a reduction in exploratory behaviour in the open arms of the EPM. The low-dose morphine (1 mg kg−1) was able to reverse mechanical hyperalgesia on day 3 and day 8 after incision, but had no effect on anxiety behaviour.

Behavioural signature of anxiety in the rat model of postoperative pain

Nociceptive and affect-related inputs from the spinal cord converge to a specific region of the amygdala and then project to several brain areas.21 The proximity of nociceptive and affect-related processing in the brain suggests their close association. This suggestion is unequivocally supported by clinical evidence, which indicates that pain and anxiety are closely related conditions.22

The present data show that acute postoperative pain by plantar incision has an anxiogenic effect in rats. The findings of this experiment are in agreement with clinical studies that have shown how acute untreated postsurgical pain can lead to a wide range of adverse effects, including anxiety and depression.3,22,23 In this study, the animals’ initial demonstration of, and subsequent recovery from, light avoidance behaviour paralleled their hyperalgesia-like behaviour. However, EPM measures showed different results. A previous study has shown that the EPM is based on the exploratory behaviour of rodents, whereas the light–dark test is based on the rodents’ locomotor behaviour and fear of novelty.24

The dose of morphine used in this study (1 mg kg−1) was sufficient to reduce plantar incision-induced mechanical hyperalgesia on the 3rd and 8th postoperative days. Morphine, however, failed to reverse anxiety-like behaviour at any point throughout 2 days of testing. Higher doses of morphine (3 mg kg−1) used in neuropathic pain models10,14 have been shown to reverse both anxiety and pain hypersensitivity. The current study, however, used the minimum dose of morphine that was able to reduce incision-induced mechanical hyperalgesia without sedative effects (Fig. 3b). It is possible that the doses of morphine required for reducing anxiolytic behaviour are higher than the doses required to reduce mechanical hyperalgesia. Supporting this, a previous study showed that injections of morphine (4, 5, 6 and 7 mg kg−1) given 30 min before testing can significantly increase the animals’ exploration time on the EPM's open arms.25 Moreover, it remains unclear whether short-term pain relief (∼30 to 60 min before testing) could reverse associated anxiety within the animal models.

Possible significance of this study

Several operant behavioural tests have been developed in recent years to evaluate thermal and mechanical sensitivity.26,27 In these tests, animals are given a choice to escape from a compartment in which they receive nociceptive stimulation, either by repetitive mechanical stimulation with von Frey hairs,26 or thermal stimulation with a hot/cold plate.27 In these assays, animals choose between a pain compartment and a brightly lit compartment; the escape duration, or time spent in the pain compartment, is compared between control and the experimental pain models. If however, experimental manipulation such as plantar incision (current study), inflammation12 or nerve ligation12 alters light aversion behaviour, the test results could be influenced. Therefore, experiments should have a control measure without nociceptive stimulation in each group of animals. The test results may be interpreted as the percentage increase or decrease from the control experiments.

Future perspective

In an elegant review, Deumens et al.28 have shown that anxiety is an important process that is involved in the transformation of acute to chronic pain, or the development of intense acute postoperative pain. However, preclinical studies focusing on acute pain-related anxiety behaviour are lacking.7 Using the current model, future studies may define mechanisms associated with anxiety and acute postoperative pain.

Acknowledgements relating to this article

Assistance with the study: the authors thank Jasenka Borzan and an anonymous reviewer of EJA for comments on their article.

Financial support and sponsorship: this work was supported by the New Jersey Neuroscience Institute and JFK Medical Center.

Conflicts of interest: none.

Presentation: the preliminary report of this study was presented at the American Pain Society meeting at Baltimore, Maryland, USA in 2010.29 While the manuscript was under preparation, Li et al.30 (2010) published an identical observation in the ‘Pain Research and Treatment’ journal.


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