• Amygdala;
  • astrocyte;
  • cytokine;
  • depression;
  • microglia;
  • neuroinflammation;
  • olfactory bulbectomy;
  • pain;
  • peripheral nerve injury;
  • spinal nerve ligation


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

The association between chronic pain and depression is widely recognized, the comorbidity of which leads to a heavier disease burden, increased disability and poor treatment response. This study examined nociceptive responding to mechanical and thermal stimuli prior to and following L5-L6 spinal nerve ligation (SNL), a model of neuropathic pain, in the olfactory bulbectomized (OB) rat model of depression. Associated changes in the expression of genes encoding for markers of glial activation and cytokines were subsequently examined in the amygdala, a key brain region for the modulation of emotion and pain. The OB rats exhibited mechanical and cold allodynia, but not heat hyperalgesia, when compared with sham-operated counterparts. Spinal nerve ligation induced characteristic mechanical and cold allodynia in the ipsilateral hindpaw of both sham and OB rats. The OB rats exhibited a reduced latency and number of responses to an innocuous cold stimulus following SNL, an effect positively correlated with interleukin (IL)-6 and IL-10 mRNA expression in the amygdala, respectively. Spinal nerve ligation reduced IL-6 and increased IL-10 expression in the amygdala of sham rats. The expression of CD11b (cluster of differentiation molecule 11b) and GFAP (glial fibrillary acidic protein), indicative of microglial and astrocyte activation, and IL-1β in the amygdala was enhanced in OB animals when compared with sham counterparts, an effect not observed following SNL. This study shows that neuropathic pain-related responding to an innocuous cold stimulus is altered in an animal model of depression, effects accompanied by changes in the expression of neuroinflammatory genes in the amygdala.

Clinical comorbidity of depression and pain is widely recognized, with over 50% of chronic pain patients experiencing depression, while patients with depression are over twice as likely to develop chronic pain (Bair et al. 2003; Gameroff & Olfson 2006). Animal models provide an important means of understanding the neurobiological basis of depression–pain comorbidity. Depressive-like behaviour has been observed in several animal models of neuropathic pain (Fukuhara et al. 2011; Hu et al. 2009; Suzuki et al. 2007; Wang et al. 2011). Conversely, reserpine-induced monoamine depletion in rats elicits both depressive-like behaviour and mechanical allodynia (Arora et al. 2011; Nagakura et al. 2009). Chronic mild stress, chronic restraint stress and Wistar-Kyoto rat models of depression exhibit hyperalgesia to formalin and complete Freund's adjuvant, models of persistent inflammatory pain (Bardin et al. 2009; Shi et al. 2010b; Wang et al. 2012). Furthermore, mechanical allodynia following peripheral nerve injury is enhanced in Wistar-Kyoto rats (Zeng et al. 2008) and following chronic restraint stress (Norman et al. 2010b) or social isolation (Norman et al. 2010a). The olfactory bulbectomized (OB) rodent is a well-validated animal model of depression (Willner & Mitchell 2002), which exhibits behavioural, neurotransmitter, neuroendocrine and immune changes resembling those reported clinically (Kelly et al. 1997; Song & Leonard 2005). The OB-induced behavioural changes include anhedonia, decreased social behaviour, learning and memory deficits, novelty-induced hyperactivity and reduced sexual behaviour, which are selectively reversed by chronic, but not acute, antidepressant treatment (Kelly et al. 1997; Song & Leonard 2005). Recent studies have shown that OB rats exhibit mechanical allodynia, enhanced formalin-evoked inflammatory pain (Burke et al. 2010; Su et al. 2010) and increased pain responding to electrical stimulation of the dura mater (Liang et al. 2011). However, neuropathic pain responding has not been evaluated in the model, and therefore, the effect of bulbectomy on nociceptive responding to mechanical and thermal stimuli prior to and following spinal nerve ligation (SNL) was evaluated in this study.

Neuroinflammatory processes are well recognized to play important roles in the pathophysiology of both depression and chronic pain (Miller et al. 2009; Panigada & Gosselin 2011; Watkins & Maier 2005). For example, central interleukin-1 beta (IL-1β) plays a key role in chronic stress-induced depressive behaviour (Goshen et al. 2008; Koo & Duman 2008), IL-6 in the amygdala increases immobility in the forced swim test (Wu & Lin 2008), chronic stress exposure increases IL-1β production specifically in the amygdala (Porterfield et al. 2012) and repeated psychosocial stress increases microglial activation in the amygdala (Wohleb et al. 2011). Spinal inflammatory processes are essential for the development of neuropathic pain (Vallejo et al. 2010); however, the role of neuroimmune mediators in supraspinal sites such as the amygdala is less understood. Prostaglandin E2 production is increased in the amygdala in a postoperative pain model (Shavit et al. 2006), and recent data have indicated a role for tumour necrosis factor alpha (TNFα) in the amygdala in anxiety and persistent inflammatory pain (Chen et al. 2013). Reserpine-induced depression–pain syndrome is associated with enhanced central inflammatory cytokines (Arora et al. 2011) and central administration of IL-1ra ameliorates the effects of neuropathic pain on depressive behaviour (Norman et al. 2010b). Thus, glial activation and cytokines in key brain regions such as the amygdala are involved in both affective and nociceptive processing, and may be responsible for the altered nociceptive responding associated with depression. As such, a further aim of this study was to determine if interactions between OB and SNL at a behavioural level are associated with concomitant alterations in the expression of genes encoding for neuroimmune mediators in the amygdala.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Animal husbandry

Male Sprague-Dawley rats (Charles River, Margate, UK) weighing 175–200 g on arrival were singly housed in plastic-bottomed cages (45 × 25 × 20 cm) containing wood shavings as bedding, in a temperature-controlled room (21 ± 1°C), with a 12:12 h light–dark cycle (lights on at 0700 h). Rats were singly housed and fed a standard laboratory diet; food and water were available ad libitum. Baseline testing began 5 days following arrival of rats to the unit and all testing was carried out during the light phase. The experimental protocol was carried out in accordance with the guidelines and approval of the Animal Care and Research Ethics Committee, National University of Ireland, Galway, under licence from the Irish Department of Health and Children and in compliance with the European Communities Council directive 86/609. All efforts were made to minimize the number of animals used, and their suffering.

Experimental design

The experimental design is presented in Fig. 1. Essentially, animals were tested with the open field, von Frey test, Hargreaves test and the acetone drop test in order to determine baseline locomotor and nociceptive responding, following which animals were randomly assigned to either sham or OB surgery groups. Two weeks following surgery, animals were retested using the aforementioned tests. Animals were subsequently allocated to one of four groups: sham-non-SNL (sham-NSNL) (n = 8), OB-NSNL (n = 7), sham-SNL (n = 10) and OB-SNL (n = 11). Mechanical allodynia was examined on days 1, 5, 8, 12 and 15 following SNL or NSNL surgery and locomotor activity was re-examined on day 14. Animals were tested with the acetone drop test and Hargreaves test on days 19 and 20 after SNL or NSNL, respectively. Twenty-four hours following the last behavioural assessment, animals were sacrificed by decapitation and the amygdala dissected out rapidly on an ice-cold plate and stored at −80°C until quantitative real-time polymerase chain reaction (RT-PCR) analysis was performed for the expression of inflammatory mediators. The genes selected for analysis included cluster of differentiation molecule 11b (CD11b, a marker of microglial activation); glial fibrillary acidic protein (GFAP, a marker of astrocyte activation); the proinflammatory cytokines, IL-1β, IL-6 and TNFα and the anti-inflammatory cytokine IL-10.


Figure 1. Experimental protocol. Abbreviations: OB, olfactory bulbectomy; SNL, spinal nerve ligation; NSNL, non-spinal nerve ligation.

Download figure to PowerPoint

Olfactory bulbectomy surgery

Bilateral OB was performed on rats anaesthetized with isoflurane [Abbot Laboratories, Berkshire, UK (3% induction, 1.5% maintenance in 0.5 l/min O2)] as outlined previously (Burke et al. 2010; Roche et al. 2007, 2008). In brief, following the application of local anaesthetic (bupivacaine HCl 0.25%), the head was shaved and a midline sagittal incision was made in the skin overlying the skull. Two burr holes of 2-mm diameter were drilled into the skull 5 mm rostral to bregma and 2 mm lateral to the midline. The olfactory bulbs were removed by gentle aspiration with a blunt hypodermic needle attached to a water vacuum pump and care was taken not to damage the frontal cortex. The burr holes were then plugged with a haemostatic sponge (Lohans Pharmacy, Galway, Ireland) to control bleeding. Sham-operated animals were treated in the same manner but the bulbs were left intact. Animals were handled daily following surgery and lesions were verified by gross inspection after completion of the study. Animals were eliminated from the analysis if the bulbs were not completely removed or if damage extended to the frontal cortex. Sham-operated animals were removed from the analysis if there was any damage to the bulbs or the frontal cortex.

L5-L6 SNL surgery

L5-L6 SNL is a well-characterized model of chronic neuropathic pain and was carried out as described previously (Kim & Chung 1992; Moriarty et al. 2012). Briefly, the rats were anaesthetized with isoflurane (2.5% in 0.6 l/min O2), the fur lateral to the midline on the left-hand side at the lower lumbar and sacral regions was clipped and an incision was made through the skin between the spinal column and the left iliac crest. Paraspinal muscles were removed using a toothed forceps to visualize the L6 transverse process, which was removed and the L5 and L6 nerves were tightly ligated using 6-0 silk suture (Interfocus, Cambridge, UK). The NSNL rats were treated in the same manner; however, the L5 and L6 nerves were exposed but not ligated. Rats were allowed to recover from anaesthesia in heated recovery cages and subsequently returned to their home cage.

Behavioural testing

Open field test

Open field behavioural testing was carried out the day prior to sham or OB surgery, on day 14 after sham/bulbectomy and on day 14 after SNL/NSNL surgery. Exposure of OB rats to a novel open field arena results in a hyperactive locomotor response, a hallmark of depressive-like behaviour in the model that is selectively reversed by chronic, but not acute, antidepressant treatment (Kelly et al. 1997; Song & Leonard 2005). As such, locomotor activity was assessed over a 5-min period in the open field to confirm OB-induced hyperactivity 14 days after surgery and to confirm that the depressive-like phenotype was maintained following SNL/NSNL surgery. On the experimental day, each animal was removed from the home cage during the light phase between 1000 h and 1200 h and placed singly into a brightly lit (lux 300–400) novel open field arena (diameter 75 cm), where locomotor activity (distance moved, cm) and time spent (seconds) in the inner zone (diameter 55 cm) were assessed using a computerized video tracking system (EthoVision®, Version 3.1, Noldus, Wageningen, the Netherlands).

von Frey test

The arena used for von Frey testing consisted of a six-compartment Perspex arena (11 × 20 × 15 cm) with wire mesh flooring. Rats were habituated to the arena for 20 min prior to testing, following which von Frey filaments (Touch-Test® Sensory Evaluators, North Coast Medical, Inc., Gilroy, CA, USA) of different forces (0.16–100 g) were used to determine the 50% withdrawal threshold as previously described (Moriarty et al. 2012). Briefly, filaments were applied perpendicular to the plantar surface of the hindpaw, with sufficient force to cause slight buckling of the filament, for up to a maximum of 5 seconds or until flinching, licking or withdrawal of the paw occurred. Filaments of increasing force were applied to both left and right hindpaws five times (alternating between paws) until a 100% positive response (five positive responses to five applications) was observed. The filament force eliciting a 50% response was calculated by plotting the percentage response vs. filament force for each rat. von Frey testing was carried out prior to and 17 days following OB/sham surgery and on days 1, 5, 8, 12 and 15 after SNL/NSNL surgery by an experimenter blind to the treatment procedure.

Acetone drop test

The acetone drop test was used to measure responding to an innocuous cold stimulus as previously described (Moriarty et al. 2012). Animals were placed in individual chambers on an elevated mesh floor and allowed to habituate for 20 min as for the von Frey test. Polyethylene tubing (2 mm ID, Fisher Scientific, Dublin, Ireland) was attached to a 1-ml syringe and used to apply approximately 0.2 ml of acetone (Sigma-Aldrich, Dublin, Ireland) to the plantar surface of the hindpaw. Latency to respond and withdrawal frequency (number of responses) within 60 seconds were recorded. A positive response was considered as a flinch, lick or withdrawal of the hindpaw. If the animal did not respond within 60 seconds, this value was taken as the latency. Each animal received eight trials in total, four per paw, alternating between left and right, with at least a 3-min interval.

Hargreaves test

The Hargreaves apparatus (Plantar Analgesia Meter, IITC Life Science Inc., Woodland Hills, CA, USA) was used to measure thermal nociception and to assess heat hyperalgesia, as previously described (Moriarty et al. 2012). Animals were placed in the apparatus on top of a glass panel heated to 30°C and allowed to habituate for 20 min. A focused beam of radiant light (active intensity of 30%) was used to heat the plantar surface of the hindpaw, and the latency to flinch, lick or withdraw the hindpaw was recorded. To prevent tissue damage, a cut-off parameter of 20 seconds was set. If no response occurred during this time, the cut-off time of 20 seconds was recorded as the latency. Each animal received eight trials in total, four per paw alternating between left and right, with at least a 3-min interval between testing.

Gene expression analysis using quantitative RT-PCR

RNA was extracted from amygdala tissue using NucleoSpin RNA II total RNA isolation kit (Macherey-Nagel, Düren, Germany). Genomic DNA contamination was removed with the addition of DNase to the samples according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using a High-Capacity cDNA Archive kit (Applied Biosystems, Warrington, UK). TaqMan gene expression assays (Applied Biosystems) containing forward and reverse primers and a FAM-labelled MGB TaqMan probe were used to quantify the gene of interest and RT-PCR was performed using an ABI Prism 7500 instrument (Applied Biosystems), as previously described (Kerr et al. 2012, 2013). Assay IDs for the genes examined were as follows: CD11b (Rn00709342_m1); GFAP (Rn00566603_m1); IL-1β (Rn00580432_m1); TNFα (Rn99999017_m1); IL-6 (Rn00561420_m1) and IL-10 (Rn00563409_m1). Polymerase chain reaction was performed using TaqMan Universal PCR Master Mix. The cycling conditions were 90°C for 10 min and 40 cycles of 90°C for 15 min followed by 60°C for 1 min. β-Actin was used as an endogenous control to normalize gene expression data. Relative gene expression was calculated using the δδCT method and data were expressed as % sham-NSNL controls.

Statistical analysis

PASW 18 statistical program was used to analyse all data. Data were assessed using either Student's unpaired t-test, Mann–Whitney U-test, two-way analysis of variance (anova) or repeated measures anova to assess changes over time. Duncan's post hoc analysis was performed following anova where appropriate. Spearman's correlation analysis was used to assess the correlation between behavioural data and gene expression data. The level of significance was set at P ≤ 0.05. All data were expressed as means ± standard error of the mean (SEM).


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Spinal nerve ligation induces anxiety-related behaviour in sham but not OB rats

The OB animals showed a characteristic hyperactivity response on exposure to the open field test 14 days following surgery, expressed as an increase in distance moved, when compared with sham-operated counterparts (t34 = 5.02, P < 0.001; Fig. 2a), an effect maintained following NSNL or SNL surgery (two-way anova effect of OB: F1,32 = 4.44, P = 0.043; Fig. 2b). Although OB rats exhibited a slight decrease in the amount of time spent in the inner zone, this effect failed to reach statistical significance, indicating no alteration in anxiety-related behaviour (Fig. 2b). Although SNL did not alter distance moved in the open field, it did reduce time spent in the inner zone in sham (sham-NSNL 47.53 ± 13.47 seconds vs. sham-SNL 14.75 ± 4.12 seconds; two-way anova effect of SNL: F1,31 = 4.83, P = 0.036; Fig. 2b), but not OB, animals when compared with NSNL controls.


Figure 2. Locomotor activity and nociceptive responding of sham and OB rats to mechanical and thermal (heat and cold) stimuli. (a) Distance moved in the open field of sham and OB animals 14 days post-surgery. (b) Distance moved and time in the inner area of the open field of sham and OB animals following NSNL or SNL surgery, (c) paw withdrawal latency and (d) withdrawal frequency (number of responses) following the application of acetone to the hindpaws, (e) mechanical withdrawal thresholds of the hindpaws to von Frey mechanical stimulation and (f) latency to withdraw from a noxious heat stimulus (Hargreaves test) applied to the hindpaws. Data expressed as mean ± SEM, n = 18–20. *P < 0.05, **P < 0.01 vs. sham-operated controls. OB, olfactory bulbectomy.

Download figure to PowerPoint

Olfactory bulbectomized rats exhibit mechanical and cold allodynia, but not heat hyperalgesia

The OB rats exhibited cold allodynia, expressed as a decrease in the latency to respond (lick, shake or withdraw the hindpaws; t34 = 2.15, P = 0.039) and a slight but non-significant increase in the withdrawal frequency (U = 103.5, P = 0.06) to the application of acetone to the hindpaws, when compared with sham-operated controls (Fig. 2b,c). In addition, OB rats displayed mechanical allodynia, showed as a significant reduction in the 50% mechanical withdrawal threshold when compared with sham-operated controls (t34 = 2.71, P = 0.011; Fig. 2d). There was no significant effect of OB on the latency to respond to a noxious heat stimulus in the Hargreaves test (Fig. 2e).

Olfactory bulbectomized rats exhibit altered SNL-induced behavioural responding to an innocuous cold stimulus

Following SNL surgery, both sham and OB rats exhibited a significant decrease in mechanical withdrawal thresholds of the ipsilateral hindpaw at all post-injury time points examined, when compared with pre-SNL thresholds (repeated measures anova effect of time F5,165 = 12.12, P < 0.001; time × side interaction F5,165 = 6.05, P < 0.001; Fig. 3a). Spinal nerve ligation did not alter the OB-induced mechanical allodynia of the contralateral (right) hindpaw (Fig. 3a; sham-contralateral vs. OB-contralateral). Because of the differences in nociceptive responding of sham and OB rats prior to SNL, behavioural data obtained following SNL were expressed as a percentage of pre-SNL values.


Figure 3. Nociceptive responding of sham and OB rats to mechanical and thermal (heat and cold) stimuli following SNL surgery. (a) Mechanical response thresholds of the ipsilateral and contralateral hindpaw of sham and OB animals prior to and following SNL surgery (**P < 0.01 Sham-ipsi and OB-ipsi vs. pre-SNL levels, ++P < 0.01 OB-ipsi/contra vs. Sham-ipsi/contra). (b) Mechanical, cold and heat responding of the ipsilateral hindpaw of sham and OB rats following SNL expressed as percentage of pre-SNL values. Mechanical responding calculated as an average over post-SNL testing period (+P < 0.05, ++P < 0.01 OB vs. sham, dotted line represents pre-SNL levels). (c) Mechanical, cold and heat responding of the contralateral hindpaw of sham and OB rats following SNL expressed as percentage of pre-SNL values. Data expressed as mean ± SEM, n = 7–11. contra, contralateral; ipsi, ipsilateral; NSNL, non-spinal nerve ligation; OB, olfactory bulbectomy; SNL, spinal nerve ligation.

Download figure to PowerPoint

SNL induced mechanical and cold allodynia, but not heat hyperalgesia, of the ipsilateral hindpaw of both sham and OB rats (Fig. 3b,c when compared with the dotted line representing pre-SNL levels). OB rats exhibited reduced paw withdrawal latency (t20 = 2.76, P = 0.012) and withdrawal frequency (t19 = 3.21, P = 0.005) to application of acetone to the ipsilateral hindpaw following SNL, when compared with sham-SNL controls, indicating altered nociceptive responding to a cold stimulus (OB-SNL vs. sham-SNL, Fig. 3b). There was no significant difference between sham and OB rats in the response threshold to mechanical, cold or heat stimuli of the contralateral hindpaw following SNL (Fig. 3c).

Olfactory bulbectomy and SNL induced changes in gene expression of inflammatory mediators in the amygdala

Analysis of inflammatory mediator expression in the right and left amygdala showed no significant lateralization effects, thus data were pooled for subsequent analysis. Olfactory bulbectomy resulted in increased mRNA expression of CD11b (marker of microglial activation), GFAP (marker of astrocyte activation) and the proinflammatory cytokine IL-1β in the amygdala when compared with sham counterparts (sham-NSNL vs. OB-NSNL, Fig. 4a–c), an effect attenuated in the presence of SNL (OB-NSNL vs. OB-SNL; two-way anova OB × SNL interactions CD11b: F1,27 = 4.22, P = 0.05; GFAP: F1,27 = 5.77, P = 0.023; IL-1β: F1,24 = 4.50, P = 0.04). There was no effect of OB or SNL on TNFα expression; however, SNL resulted in reduced expression of IL-6 (SNL: F1,23 = 21.4, P < 0.01) in both sham and OB animals, and increased expression of IL-10 (SNL: F1,24 = 10.56, P = 0.003) in sham, but not OB, animals (Fig. 4e,f).


Figure 4. Gene expression of neuroinflammatory mediators in the amygdala following OB and SNL. The effect of OB, SNL and OB-SNL on the expression of (a) CD11b, (b) GFAP, (c) IL-1β, (d) TNFα, (e) IL-6 and (f) IL-10. Data expressed as mean ± SEM, n = 7–8. *P < 0.05, **P < 0.01 vs. sham-NSNL, #P < 0.05 vs. OB-NSNL, (g) significant correlation between IL-6 expression and latency to respond to the cold stimulus post SNL, (h) significant correlation between IL-10 expression and withdrawal frequency to the cold innocuous stimulus post SNL. CD11b, cluster of differentiation molecule 11b; GFAP, glial fibrillary acidic protein; IL-1β, interleukin-1 beta; NSNL, non-spinal nerve ligation; OB, olfactory bulbectomy; SNL, spinal nerve ligation; TNFα, tumour necrosis factor alpha.

Download figure to PowerPoint

Spearman's correlation analysis showed a significant positive correlation between paw withdrawal latency in the acetone drop test following SNL surgery and IL-6 mRNA expression in the amygdala (r = 0.620, P = 0.006, Fig. 4g). The percentage change in withdrawal frequency of the ipsilateral hindpaw in the acetone drop test following SNL surgery was positively correlated with IL-10 expression in the amygdala (r = 0.381, P = 0.046, Fig. 4h). There were no further significant correlations between behavioural responding and gene expression of inflammatory mediators.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

This study shows that the OB rat model of depression displays mechanical and cold allodynia, and altered neuropathic pain behaviour to an innocuous cold stimulus following SNL surgery. The reduced latency and increased number of responses in the acetone drop test following SNL surgery were positively correlated with IL-6 and IL-10 expression in the amygdala, respectively. Furthermore, the OB-associated increase in the expression of CD11b, GFAP and IL-1β in the amygdala was not observed in SNL animals. This study provides a further preclinical model for studying the association between depression and neuropathic pain, and indicates that neuroimmune processes in the amygdala may, in part, underlie the behavioural changes observed.

Altered nociceptive responding in the OB model of depression

Prior to spinal nerve injury, OB rats exhibited mechanical allodynia, results consistent with those previously reported from our laboratory (Burke et al. 2010); however, to our knowledge, this is the first study to report that OB rats also exhibit cold allodynia. In comparison, nociceptive responding to heat stimuli was not altered in OB rats in this study, although increased paw withdrawal latency to a radiant heat source and transient thermal heat hyperalgesia in the hot plate, but not tail flick, test have been previously reported in OB rats (Burke et al. 2010; Su et al. 2010; Wang et al. 2010). Thus, OB animals appear to exhibit alterations in nociceptive responding to thermal stimuli depending on the test employed. The present findings correlate with those observed clinically where depressed patients exhibit decreased (Bar et al. 2007; Lautenbacher et al. 1994; Schwier et al. 2010), increased (Chiu et al. 2005; Gormsen et al. 2004; Strigo et al. 2008) and no change (Graff-Guerrero et al. 2008) in sensitivity to experimental pain, depending on the modality and intensity of the stimulus.

This study is the first to examine the effect of bulbectomy on neuropathic pain responding, showing that OB rats exhibit altered nociceptive responding to an innocuous cold, but not heat or mechanical, stimulus following SNL. Specifically, OB-SNL rats exhibited reduced latency to respond and reduced number of responses of the ipsilateral hindpaw to an innocuous cold stimulus. We propose that the reduction in response latency may reflect enhanced initial perception of cold stimuli, whereas the reduction in the number of responses might reflect a concomitant reduction in the duration or magnitude of the response in OB-SNL animals. The mechanisms mediating these alterations remain to be determined; however, it is possible that bulbectomy may result in disruption of nociceptive gating and descending pain pathways. Thus, following nerve injury, OB animals may exhibit enhanced gating of cold stimulus-related nociceptive information (reduced latency); however, following initial perception, OB animals may be capable of engaging the descending inhibitory pain pathway more effectively, resulting in reduced magnitude or severity (number of responses) to the stimulus. It is possible that functional alterations in key brain regions part of the descending pain pathway such as the amygdala, and changes in glutamatergic, serotonergic or noradrenergic neurotransmission (Kelly et al. 1997; Song & Leonard 2005) may underlie the enhanced initial perception and/or the decrease in the magnitude of the SNL-induced cold allodynia in OB rats. This idea is discussed further below in the context of the OB-related changes in IL-6 and IL-10 expression observed herein. In contrast to our findings here, SNL and partial sciatic ligation result in a paradoxical increase in mechanical and thermal (heat) thresholds in the unpredictable chronic mild stress (Shi et al. 2010a) and the Flinders sensitive line (Shir et al. 2001) models of depression, respectively. Thus, differential neurobiological mechanisms may underlie the effects of unpredictable chronic mild stress and OB on nociceptive responding following SNL.

In accordance with previous studies (Suzuki et al. 2007; Yalcin et al. 2011), SNL induced anxiety-related behaviour in sham rats in the open field test, exemplified by the reduction in time in the inner area. In comparison, OB-SNL rats did not exhibit anxiety-related behaviour but maintained the OB-induced increase in locomotor activity on exposure to the open field. Although it cannot be ruled out that SNL may induce anxiety-related behaviour in OB animals at times or in paradigms other than those examined in this study, the present data indicate that OB rats exhibit resilience to SNL-induced anxiogenesis in the open field.

Gene expression changes in the amygdala associated with altered nociceptive responding in the OB model

Limbic regions such as the amygdala play key roles in the processing of emotion and pain, and inflammatory mediators in discrete brain regions modulate both affective and nociceptive processing. Thus, neuroimmune processes in regions such as the amygdala may be the driving force for altered nociceptive responding associated with depression. Previous studies have shown that OB rats exhibit increases in TNFα and/or IL-1β levels in the prefrontal cortex, hippocampus and hypothalamus (Borre et al. 2012; Myint et al. 2007), increased phospholipase A2 and prostaglandin E2 in the hypothalamus (Song et al. 2009) and increased GFAP in the frontal cortex (Cizkova et al. 1997). To our knowledge, this is the first study to investigate the effect of bulbectomy on the expression of immune mediators in the amygdala, showing enhanced gene expression of CD11b and GFAP, markers of microglial and astrocyte activation, respectively, and of the proinflammatory cytokine IL-1β in the amygdala of OB rats. OB-induced hyperactivity has been shown to be associated with neurodegeneration within the amygdala (Jarosik et al. 2007; Wrynn et al. 2000), effects that may result from neuroinflammatory processes in this region following removal of the bulbs. In addition, increased glial activation and IL-1β within the amygdala may be responsible for OB-induced mechanical and cold allodynia, as it has been shown that intracerebroventricular administration of non-pyrogenic doses of IL-1β results in thermal (Oka et al. 1993) and mechanical (Yabuuchi et al. 1996) hyperalgesia. Interestingly, the increase in the expression of IL-1β in the amygdala of OB rats was not observed following SNL, suggesting that spinal nerve injury can attenuate the increases in amygdaloid IL-1β expression that result from central injury (OB). Indeed, SNL induced a decrease in IL-6 and an increase in IL-10 expression in the amygdala. The lack of effect of SNL on the expression of CD11b, GFAP or other proinflammatory cytokines (IL-1β and TNFα) may not be surprising given the time at which these were examined post-surgery (day 22). Previous studies have shown that GFAP, IL-1β and TNFα protein levels are increased in the brain between days 3 and 7 post-surgery (Liu et al. 2007; Marcello et al. 2013; Xie et al. 2006). In comparison, IL-10 protein levels in the brain have been reported to increase over time following SNL, with highest levels observed 21 days post-surgery (Xie et al. 2006), correlating with the present findings.

Latency to respond to a cold thermal stimulus was positively correlated with IL-6 expression in the amygdala; thus, enhanced initial cold perception observed in OB animals is associated with, and possibly mediated by, low IL-6 levels in the amygdala. The number of responses to the innocuous cold stimulus was positively correlated with IL-10 expression in the amygdala. As mentioned, IL-10 levels in the brain have been reported to be enhanced following SNL (Xie et al. 2006), although region-specific changes have not been investigated. Pharmacological and genetic deletion of IL-10 is associated with depressive-like behaviour (Mesquita et al. 2008) and increased thermal nociceptive thresholds (Tu et al. 2003). Interleukin-10 is known to protect against glial and neuronal cell death (Bachis et al. 2001; Strle et al. 2002), and thus changes in IL-10 expression may alter neuronal integrity and function within the amygdala. Such changes may increase descending inhibitory pain regulation and/or reduce descending facilitatory pain modulation, resulting in reduced magnitude or severity (number of responses) to the cold stimulus as observed in OB-SNL rats.

In conclusion, the present findings show that in the presence of a depressive-like phenotype, neuropathic pain behaviour is altered depending on stimulus modality, effects accompanied by alterations in inflammatory mediator gene expression within the amygdala. Increased understanding of the neurobiological substrates underlying depression, chronic pain and the interaction between these disorders may provide novel therapeutic targets for treating these debilitating comorbid disorders.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  • Arora, V., Kuhad, A., Tiwari, V. & Chopra, K. (2011) Curcumin ameliorates reserpine-induced pain-depression dyad: behavioural, biochemical, neurochemical and molecular evidences. Psychoneuroendocrinology 36, 15701581.
  • Bachis, A., Colangelo, A.M., Vicini, S., Doe, P.P., De Bernardi, M.A., Brooker, G. & Mocchetti, I. (2001) Interleukin-10 prevents glutamate-mediated cerebellar granule cell death by blocking caspase-3-like activity. J Neurosci 21, 31043112.
  • Bair, M.J., Robinson, R.L., Katon, W. & Kroenke, K. (2003) Depression and pain comorbidity: a literature review. Arch Intern Med 163, 24332445.
  • Bar, K.J., Wagner, G., Koschke, M., Boettger, S., Boettger, M.K., Schlosser, R. & Sauer, H. (2007) Increased prefrontal activation during pain perception in major depression. Biol Psychiatry 62, 12811287.
  • Bardin, L., Malfetes, N., Newman-Tancredi, A. & Depoortere, R. (2009) Chronic restraint stress induces mechanical and cold allodynia, and enhances inflammatory pain in rat: relevance to human stress-associated painful pathologies. Behav Brain Res 205, 360366.
  • Borre, Y., Sir, V., de Kivit, S., Westphal, K.G., Olivier, B. & Oosting, R.S. (2012) Minocycline restores spatial but not fear memory in olfactory bulbectomized rats. Eur J Pharmacol 697, 5964.
  • Burke, N.N., Hayes, E., Calpin, P., Kerr, D.M., Moriarty, O., Finn, D.P. & Roche, M. (2010) Enhanced nociceptive responding in two rat models of depression is associated with alterations in monoamine levels in discrete brain regions. Neuroscience 171, 13001313.
  • Chen, J., Song, Y., Yang, J., Zhang, Y., Zhao, P., Zhu, X.J. & Su, H.C. (2013) The contribution of TNF-alpha in the amygdala to anxiety in mice with persistent inflammatory pain. Neurosci Lett 541, 275280.
  • Chiu, Y.H., Silman, A.J., Macfarlane, G.J., Ray, D., Gupta, A., Dickens, C., Morriss, R. & McBeth, J. (2005) Poor sleep and depression are independently associated with a reduced pain threshold. Results of a population based study. Pain 115, 316321.
  • Cizkova, D., Racekova, E. & Vanicky, I. (1997) The expression of B-50/GAP-43 and GFAP after bilateral olfactory bulbectomy in rats. Physiol Res 46, 487495.
  • Fukuhara, K., Ishikawa, K., Yasuda, S., Kishishita, Y., Kim, H.K., Kakeda, T., Yamamoto, M., Norii, T. & Ishikawa, T. (2011) Intracerebroventricular 4-methylcatechol (4-MC) ameliorates chronic pain associated with depression-like behavior via induction of brain-derived neurotrophic factor (BDNF). Cell Mol Neurobiol 32, 971977.
  • Gameroff, M.J. & Olfson, M. (2006) Major depressive disorder, somatic pain, and health care costs in an urban primary care practice. J Clin Psychiatry 67, 12321239.
  • Gormsen, L., Ribe, A.R., Raun, P., Rosenberg, R., Videbech, P., Vestergaard, P., Bach, F.W. & Jensen, T.S. (2004) Pain thresholds during and after treatment of severe depression with electroconvulsive therapy. Eur J Pain 8, 487493.
  • Goshen, I., Kreisel, T., Ben-Menachem-Zidon, O., Licht, T., Weidenfeld, J., Ben-Hur, T. & Yirmiya, R. (2008) Brain interleukin-1 mediates chronic stress-induced depression in mice via adrenocortical activation and hippocampal neurogenesis suppression. Mol Psychiatry 13, 717728.
  • Graff-Guerrero, A., Pellicer, F., Mendoza-Espinosa, Y., Martinez-Medina, P., Romero-Romo, J. & de la Fuente-Sandoval, C. (2008) Cerebral blood flow changes associated with experimental pain stimulation in patients with major depression. J Affect Disord 107, 161168.
  • Hu, B., Doods, H., Treede, R.D. & Ceci, A. (2009) Depression-like behaviour in rats with mononeuropathy is reduced by the CB2-selective agonist GW405833. Pain 143, 206212.
  • Jarosik, J., Legutko, B., Unsicker, K. & von Bohlen Und Halbach, O. (2007) Antidepressant-mediated reversal of abnormal behavior and neurodegeneration in mice following olfactory bulbectomy. Exp Neurol 204, 2028.
  • Kelly, J.P., Wrynn, A.S. & Leonard, B.E. (1997) The olfactory bulbectomized rat as a model of depression: an update. Pharmacol Ther 74, 299316.
  • Kerr, D.M., Burke, N.N., Ford, G.K., Connor, T.J., Harhen, B., Egan, L.J., Finn, D.P. & Roche, M. (2012) Pharmacological inhibition of endocannabinoid degradation modulates the expression of inflammatory mediators in the hypothalamus following an immunological stressor. Neuroscience 1, 5363.
  • Kerr, D.M., Harhan, B., Okine, B.N., Egan, L.J., Finn, D.P. & Roche, M. (2013) The monoacylglycerol lipase inhibitor JZL184 attenuates LPS-induced increases in cytokine expression in the rat frontal cortex and plasma: differential mechanisms of action. Br J Pharmacol 169, 808819.
  • Kim, S.H. & Chung, J.M. (1992) An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 50, 355363.
  • Koo, J.W. & Duman, R.S. (2008) IL-1beta is an essential mediator of the antineurogenic and anhedonic effects of stress. Proc Natl Acad Sci U S A 105, 751756.
  • Lautenbacher, S., Roscher, S., Strian, D., Fassbender, K., Krumrey, K. & Krieg, J.C. (1994) Pain perception in depression: relationships to symptomatology and naloxone-sensitive mechanisms. Psychosom Med 56, 345352.
  • Liang, J., Yu, S., Dong, Z., Wang, X., Liu, R., Chen, X. & Li, Z. (2011) The effects of OB-induced depression on nociceptive behaviors induced by electrical stimulation of the dura mater surrounding the superior sagittal sinus. Brain Res 1424, 919.
  • Liu, J., Feng, X., Yu, M., Xie, W., Zhao, X., Li, W., Guan, R. & Xu, J. (2007) Pentoxifylline attenuates the development of hyperalgesia in a rat model of neuropathic pain. Neurosci Lett 412, 268272.
  • Marcello, L., Cavaliere, C., Colangelo, A.M., Bianco, M.R., Cirillo, G., Alberghina, L. & Papa, M. (2013) Remodelling of supraspinal neuroglial network in neuropathic pain is featured by a reactive gliosis of the nociceptive amygdala. Eur J Pain 17, 799810.
  • Mesquita, A.R., Correia-Neves, M., Roque, S., Castro, A.G., Vieira, P., Pedrosa, J., Palha, J.A. & Sousa, N. (2008) IL-10 modulates depressive-like behavior. J Psychiatr Res 43, 8997.
  • Miller, A.H., Maletic, V. & Raison, C.L. (2009) Inflammation and its discontents: the role of cytokines in the pathophysiology of major depression. Biol Psychiatry 65, 732741.
  • Moriarty, O., Roche, M., McGuire, B.E. & Finn, D.P. (2012) Validation of an air-puff passive-avoidance paradigm for assessment of aversive learning and memory in rat models of chronic pain. J Neurosci Methods 204, 18.
  • Myint, A.M., Steinbusch, H.W., Goeghegan, L., Luchtman, D., Kim, Y.K. & Leonard, B.E. (2007) Effect of the COX-2 inhibitor celecoxib on behavioural and immune changes in an olfactory bulbectomised rat model of depression. Neuroimmunomodulation 14, 6571.
  • Nagakura, Y., Oe, T., Aoki, T. & Matsuoka, N. (2009) Biogenic amine depletion causes chronic muscular pain and tactile allodynia accompanied by depression: a putative animal model of fibromyalgia. Pain 146, 2633.
  • Norman, G.J., Karelina, K., Morris, J.S., Zhang, N., Cochran, M. & Courtney DeVries, A. (2010a) Social interaction prevents the development of depressive-like behavior post nerve injury in mice: a potential role for oxytocin. Psychosom Med 72, 519526.
  • Norman, G.J., Karelina, K., Zhang, N., Walton, J.C., Morris, J.S. & Devries, A.C. (2010b) Stress and IL-1beta contribute to the development of depressive-like behavior following peripheral nerve injury. Mol Psychiatry 15, 404414.
  • Oka, T., Aou, S. & Hori, T. (1993) Intracerebroventricular injection of interleukin-1 beta induces hyperalgesia in rats. Brain Res 624, 6168.
  • Panigada, T. & Gosselin, R.D. (2011) Behavioural alteration in chronic pain: are brain glia involved? Med Hypotheses 77, 584588.
  • Porterfield, V.M., Gabella, K.M., Simmons, M.A. & Johnson, J.D. (2012) Repeated stressor exposure regionally enhances beta-adrenergic receptor-mediated brain IL-1beta production. Brain Behav Immun 26, 12491255.
  • Roche, M., Harkin, A. & Kelly, J.P. (2007) Chronic fluoxetine treatment attenuates stressor-induced changes in temperature, heart rate, and neuronal activation in the olfactory bulbectomized rat. Neuropsychopharmacology 32, 13121320.
  • Roche, M., Shanahan, E., Harkin, A. & Kelly, J.P. (2008) Trans-species assessment of antidepressant activity in a rodent model of depression. Pharmacol Rep 60, 404408.
  • Schwier, C., Kliem, A., Boettger, M.K. & Bar, K.J. (2010) Increased cold-pain thresholds in major depression. J Pain 11, 287290.
  • Shavit, Y., Fridel, K. & Beilin, B. (2006) Postoperative pain management and proinflammatory cytokines: animal and human studies. J Neuroimmune Pharmacol 1, 443451.
  • Shi, M., Qi, W.J., Gao, G., Wang, J.Y. & Luo, F. (2010a) Increased thermal and mechanical nociceptive thresholds in rats with depressive-like behaviors. Brain Res 1353, 225233.
  • Shi, M., Wang, J.Y. & Luo, F. (2010b) Depression shows divergent effects on evoked and spontaneous pain behaviors in rats. J Pain 11, 219229.
  • Shir, Y., Zeltser, R., Vatine, J.J., Carmi, G., Belfer, I., Zangen, A., Overstreet, D., Raber, P. & Seltzer, Z. (2001) Correlation of intact sensibility and neuropathic pain-related behaviors in eight inbred and outbred rat strains and selection lines. Pain 90, 7582.
  • Song, C. & Leonard, B.E. (2005) The olfactory bulbectomised rat as a model of depression. Neurosci Biobehav Rev 29, 627647.
  • Song, C., Zhang, X.Y. & Manku, M. (2009) Increased phospholipase A2 activity and inflammatory response but decreased nerve growth factor expression in the olfactory bulbectomized rat model of depression: effects of chronic ethyl-eicosapentaenoate treatment. J Neurosci 29, 1422.
  • Strigo, I.A., Simmons, A.N., Matthews, S.C., Craig, A.D. & Paulus, M.P. (2008) Association of major depressive disorder with altered functional brain response during anticipation and processing of heat pain. Arch Gen Psychiatry 65, 12751284.
  • Strle, K., Zhou, J.H., Broussard, S.R., Venters, H.D., Johnson, R.W., Freund, G.G., Dantzer, R. & Kelley, K.W. (2002) IL-10 promotes survival of microglia without activating Akt. J Neuroimmunol 122, 919.
  • Su, Y.L., Wang, N., Gao, G., Wang, J.Y. & Luo, F. (2010) The effect of depression on the thermal nociceptive thresholds in rats with spontaneous pain. Neurosci Bull 26, 429436.
  • Suzuki, T., Amata, M., Sakaue, G., Nishimura, S., Inoue, T., Shibata, M. & Mashimo, T. (2007) Experimental neuropathy in mice is associated with delayed behavioral changes related to anxiety and depression. Anesth Analg 104, 15701577.
  • Tu, H., Juelich, T., Smith, E.M., Tyring, S.K., Rady, P.L. & Hughes, T.K. Jr. (2003) Evidence for endogenous interleukin-10 during nociception. J Neuroimmunol 139, 145149.
  • Vallejo, R., Tilley, D.M., Vogel, L. & Benyamin, R. (2010) The role of glia and the immune system in the development and maintenance of neuropathic pain. Pain Pract 10, 167184.
  • Wang, W., Qi, W.J., Xu, Y., Wang, J.Y. & Luo, F. (2010) The differential effects of depression on evoked and spontaneous pain behaviors in olfactory bulbectomized rats. Neurosci Lett 472, 143147.
  • Wang, J., Goffer, Y., Xu, D., Tukey, D.S., Shamir, D.B., Eberle, S.E., Zou, A.H., Blanck, T.J. & Ziff, E.B. (2011) A single subanesthetic dose of ketamine relieves depression-like behaviors induced by neuropathic pain in rats. Anesthesiology 115, 812821.
  • Wang, S., Tian, Y., Song, L., Lim, G., Tan, Y., You, Z., Chen, L. & Mao, J. (2012) Exacerbated mechanical hyperalgesia in rats with genetically predisposed depressive behavior: role of melatonin and NMDA receptors. Pain 153, 24482457.
  • Watkins, L.R. & Maier, S.F. (2005) Immune regulation of central nervous system functions: from sickness responses to pathological pain. J Intern Med 257, 139155.
  • Willner, P. & Mitchell, P.J. (2002) The validity of animal models of predisposition to depression. Behav Pharmacol 13, 169188.
  • Wohleb, E.S., Hanke, M.L., Corona, A.W., Powell, N.D., Stiner, L.M., Bailey, M.T., Nelson, R.J., Godbout, J.P. & Sheridan, J.F. (2011) beta-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat. J Neurosci 31, 62776288.
  • Wrynn, A.S., Sebens, J.B., Koch, T., Leonard, B.E. & Korf, J. (2000) Prolonged c-Jun expression in the basolateral amygdala following bulbectomy: possible implications for antidepressant activity and time of onset. Brain Res Mol Brain Res 76, 717.
  • Wu, T.H. & Lin, C.H. (2008) IL-6 mediated alterations on immobile behavior of rats in the forced swim test via ERK1/2 activation in specific brain regions. Behav Brain Res 193, 183191.
  • Xie, W., Luo, S., Xuan, H., Chou, C., Song, G., Lv, R., Jin, Y., Li, W. & Xu, J. (2006) Betamethasone affects cerebral expressions of NF-kappaB and cytokines that correlate with pain behavior in a rat model of neuropathy. Ann Clin Lab Sci 36, 3946.
  • Yabuuchi, K., Nishiyori, A., Minami, M. & Satoh, M. (1996) Biphasic effects of intracerebroventricular interleukin-1 beta on mechanical nociception in the rat. Eur J Pharmacol 300, 5965.
  • Yalcin, I., Bohren, Y., Waltisperger, E., Sage-Ciocca, D., Yin, J.C., Freund-Mercier, M.J. & Barrot, M. (2011) A time-dependent history of mood disorders in a murine model of neuropathic pain. Biol Psychiatry 70, 946953.
  • Zeng, Q., Wang, S., Lim, G., Yang, L., Mao, J., Sung, B., Chang, Y., Lim, J.A. & Guo, G. (2008) Exacerbated mechanical allodynia in rats with depression-like behavior. Brain Res 1200, 2738.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

The authors would like to acknowledge technical assistance from Mr Ambrose O'Halloran. The authors would like to gratefully acknowledge funding received from the Discipline of Physiology and the Millennium Fund, National University of Ireland Galway. N.B. is a recipient of College of Medicine, Nursing and Health Sciences, National University of Ireland Galway, Doctoral Fellowship. The authors declare no conflict of interest.