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A preconditioning nerve lesion inhibits mechanical pain hypersensitivity following subsequent neuropathic injury

A preconditioning nerve lesion inhibits mechanical pain hypersensitivity following subsequent... Background: A preconditioning stimulus can trigger a neuroprotective phenotype in the nervous system - a preconditioning nerve lesion causes a significant increase in axonal regeneration, and cerebral preconditioning protects against subsequent ischemia. We hypothesized that a preconditioning nerve lesion induces gene/protein modifications, neuronal changes, and immune activation that may affect pain sensation following subsequent nerve injury. We examined whether a preconditioning lesion affects neuropathic pain and neuroinflammation after peripheral nerve injury. Results: We found that a preconditioning crush injury to a terminal branch of the sciatic nerve seven days before partial ligation of the sciatic nerve (PSNL; a model of neuropathic pain) induced a significant attenuation of pain hypersensitivity, particularly mechanical allodynia. A preconditioning lesion of the tibial nerve induced a long-term significant increase in paw-withdrawal threshold to mechanical stimuli and paw-withdrawal latency to thermal stimuli, after PSNL. A preconditioning lesion of the common peroneal induced a smaller but significant short-term increase in paw-withdrawal threshold to mechanical stimuli, after PSNL. There was no difference between preconditioned and unconditioned animals in neuronal damage and macrophage and T-cell infiltration into the dorsal root ganglia (DRGs) or in astrocyte and microglia activation in the spinal dorsal and ventral horns. Conclusions: These results suggest that prior exposure to a mild nerve lesion protects against adverse effects of subsequent neuropathic injury, and that this conditioning-induced inhibition of pain hypersensitivity is not dependent on neuroinflammation in DRGs and spinal cord. Identifying the underlying mechanisms may have important implications for the understanding of neuropathic pain due to nerve injury. Background (a branch of the sciatic nerve), made 2 weeks before Peripheral nerve injury often results in neuropathic pain sciatic nerve injury, causes a significant increase in axon characterized by unpleasant and persistent increases in outgrowth [2,3]. Further work has shown that regenera- pain sensitivity, including hyperalgesia and allodynia. It tion of dorsal root ganglion (DRG) central processes is well recognized that nerve lesion induces neuronal into a peripheral nerve graft and regeneration of dorsal and immunological changes and modifies gene and pro- column fibers into a spinal cord lesion site is signifi- tein expression, both in the peripheral nervous system cantly improved by performing a peripheral nerve lesion and in the spinal cord [1]. However, whether such [4-6]. Thus, a preconditioning nerve lesion appears to changes affect neuropathic pain behavior following sub- increase the intrinsic regenerative ability of central sequent injury is not known. axons by inducing molecular changes (e.g. elevation of Studies on axonal regeneration in the sciatic nerve intracellular cyclic AMP) that allow axons to overcome have shown that a conditioning lesion of the tibial nerve myelin inhibition [7]. Studies on cerebral precondition- ing have shown that a brief period of sub-lethal or mild preconditioning ischemia attenuates injury from subse- * Correspondence: gila@unsw.edu.au School of Medical Sciences, University of New South Wales, Sydney, NSW quent severe ischemia [8]. This neuroprotection is 2052 Australia achieved by promoting neuronal survival through Full list of author information is available at the end of the article © 2011 Moalem-Taylor et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 2 of 9 http://www.molecularpain.com/content/7/1/1 attenuation of several injury-inducing mechanisms (e.g. with either the contralateral side or baseline values excitotoxicity, ion/pH imbalance, oxidative stress, meta- before surgery, as indicated by a sharp decrease in paw bolic dysfunction, inflammation) and through enhance- withdrawal threshold to mechanical stimuli and paw ment of endogenous repair processes [9]. withdrawal latency to thermal stimuli (Figure 1). On the There are limited and conflicting data regarding the contralateral side to PSNL (right hindpaws), there were effects of an existing injury on the development of pain no significant differences in either the mechanical or the subsequent to a second injury. Some studies have thermal pain sensitivity between the preconditioned demonstrated that noxious cutaneous stimuli elicit a (crush-injured) and unconditioned (no crush) rats powerful and long-lasting inhibition of spinal dorsal (Figure 1 right panel). However, on the ipsilateral side horn and trigeminal convergent neurons, termed diffuse to PSNL (left hindpaws), a preconditioning lesion of the noxious inhibitory controls (DNIC) [10,11]. DNIC left tibial nerve induced a long-term significant attenua- effects are directly related to the duration of the condi- tion of both ligation-induced mechanical allodynia tioning painful stimulus [11] and predict inhibition of (Figure 1A) and thermal hyperalgesia (Figure 1B). Com- pain behaviors following prior painful insults. Indeed, pared with unconditioned rats, preconditioned rats had activation of DNIC was confirmed in rats with chronic significantly higher (1.6-2.5-fold; P < 0.01-P < 0.001) constriction injury of the sciatic nerve, demonstrating paw withdrawal thresholds to mechanical stimuli 2-19 increased inhibition by nociceptive conditioning stimuli days post-PSNL (Figure 1A) and significantly higher to the hindpaw [12]. In contrast, other studies have (1.5-1.6-fold; P <0.05-P < 0.001) paw withdrawal laten- shown that a trigeminal nerve injury significantly accel- cies to thermal stimuli 2-9 days post-PSNL (Figure 1B). erated the development of mechanical allodynia and A preconditioning lesion of the left common peroneal hyperalgesia following a chronic constriction injury of induced a smaller but significant short-term attenuation the sciatic nerve as a second injury (day 7), in Lewis but of mechanical allodynia (Figure 1C), but not thermal not Sprague-Dawley or Sabra rats [13]. Repeated injury hyperalgesia (Figure 1D). Compared with unconditioned to the lumbar nerve roots at 42 days also produced rats, preconditioned (left peroneal) rats had significantly enhanced mechanical allodynia and spinal neuroinflam- higher (1.6-1.9-fold; P < 0.05-P < 0.01) paw withdrawal mation [14]. Thus, it appears that modulation of pain thresholds to mechanical stimuli 7-12 days post-PSNL following a primary injury is greatly dependent on the (Figure 1C). These preconditioned (left peroneal) rats location and timing of injuries, the nature of stimulus, also had higher (1.2-1.4-fold) paw withdrawal latencies and the strain of animals. to thermal stimuli 7-9 days post-PSNL (Figure 1D) com- Here, using a rat model of neuropathic pain we inves- pared with unconditioned rats, but this effect was not tigated whether a preconditioning nerve lesion influ- statistically significant. A preconditioning lesion of the ences pain sensation and neuroinflammation following a right tibial nerve induced a small decrease in paw with- subsequent distant peripheral nerve injury involving the drawal thresholds to mechanical stimuli in the contralat- same dorsal root ganglia. eral side to PSNL, though these changes were not significant (Figure 1E, right). On the ipsilateral side, a Results short-term attenuation of mechanical allodynia was A preconditioning nerve lesion alters pain behaviors observed, and preconditioned (right tibial) rats had a following neuropathic injury significantly higher (1.6-fold; P <0.05) pawwithdrawal To study the effects of preconditioning on neuropathic threshold to mechanical stimuli 4 days post-PSNL (Fig- pain, we carried out a crush injury to one of the term- ure 1E). There were no differences in thermal pain sen- inal branches of the sciatic nerve or exposed the nerve sitivity between unconditioned and preconditioned branch without crush injury (sham control), 1 week (right tibial) rats on either the ipsilateral or the contral- before partial ligation of the sciatic nerve (PSNL; a ateral sides to PSNL (data not shown). model of neuropathic pain) and measured pain beha- viors for 30 days. The following groups of rats (n = 6 A preconditioning nerve lesion does not affect PSNL- animals per group) were used: (1) Left tibial nerve crush induced neuroinflammation in DRG and spinal cord injury 1 week before left PSNL; (2) Left common pero- We next examined whether a preconditioning nerve neal nerve crush injury 1 week before left PSNL; (3) lesion affects neuroinflammation 7 days after PSNL (14 Right tibial nerve crush injury 1 week before left PSNL. days after crush injury) in L4/5 DRGs and in L4-6 spinal Each of these preconditioned groups was compared to a cord segments using immunohistochemistry (n = 3 ani- control group with a relevant sham crush 1 week before mals per group). Cryosections of excised DRGs were left PSNL (unconditioned). stained for macrophages (ED1 marker) and T cells (T- Following PSNL, all unconditioned rats developed pain cell receptor marker) [15], and for damaged neurons hypersensitivity in the paw ipsilateral to PSNL compared expressing activating transcription factor 3 (ATF3) in Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 3 of 9 http://www.molecularpain.com/content/7/1/1 Ipsilateral Contralateral 30 30 Left tibial crush+PSNL Sham+PSNL 25 25 20 20 15 15 *** ** Left tibial crush+PSNL 10 *** 10 *** *** Sham+PSNL ** *** 5 5 *** 0 0 -5 -2 1 4 7 10 13 16 19 22 25 28 31 -5 -2 1 4 7 10 13 16 19 22 25 28 31 Days Days 20 20 Left tibial crush+PSNL 18 18 Sham+PSNL 16 16 14 ** 14 12 12 10 10 8 8 6 6 ** *** ** 4 4 Left tibial crush+PSNL 2 2 Sham+PSNL 0 0 -5 -2 1 4 7 10 13 16 19 22 25 28 31 -5 -2 1 4 7 10 13 16 19 22 25 28 31 Days Days 35 35 Left peroneal crush+PSNL 30 30 Sham+PSNL 25 25 20 20 15 15 10 10 Left peroneal crush+PSNL ** 5 5 Sham+PSNL 0 0 -2 1 4 7 10 13 16 19 22 25 28 31 -2 1 4 7 10 13 16 19 22 25 28 31 Days Days 20 20 18 18 Left peroneal crush+PSNL 16 16 Sham+PSNL 14 14 12 12 10 10 8 8 6 6 4 4 Left peroneal crush+PSNL 2 2 Sham+PSNL 0 0 -2 1 4 7 10 13 16 19 22 25 28 31 -2 1 4 7 10 13 16 19 22 25 28 31 Days Days E 30 30 Right tibial crush+PSNL 25 25 Sham+PSNL 20 20 15 15 10 10 * Right tibial crush+PSNL 5 5 Sham+PSNL 0 0 -5 -2 1 4 7 10 13 16 19 22 25 28 31 -5 -2 1 4 7 10 13 16 19 22 25 28 31 Days Days Figure 1 Effects of preconditioning nerve lesion on neuropathic pain behaviors due to partial ligation of the left sciatic nerve. (A-B) Tibial nerve crush injury (left) inhibits PSNL-induced mechanical and thermal pain hypersensitivity. Withdrawal thresholds to mechanical stimuli (d9-d26) (A) and withdrawal latencies to thermal stimuli (d9-d16) (B) were significantly greater in the ipsilateral left hindpaws of rats that underwent left tibial crush injury 1 week before PSNL than in rats that underwent only PSNL. No significant differences in paw withdrawal thresholds (A) and in paw withdrawal latencies (B) were observed in the contralateral right side. (C-D) Peroneal nerve crush injury (left) inhibits PSNL-induced mechanical, but not thermal, pain hypersensitivity. Withdrawal thresholds to mechanical stimuli (d14-d19) (C) were significantly greater in ipsilateral left hindpaws of rats that underwent left peroneal crush injury 1 week before PSNL than in rats that underwent only PSNL. No significant differences in paw withdrawal thresholds were observed in the contralateral right side. (D) No significant differences were observed in paw withdrawal latencies to thermal stimuli in both the ipsilateral and contralateral sides. (E) Tibial nerve crush injury (right; on the other side of the PSNL) transiently inhibits PSNL-induced mechanical pain hypersensitivity. Withdrawal thresholds to mechanical stimuli (d11) were significantly greater in ipsilateral left hindpaws of rats that underwent right tibial crush injury 1 week before PSNL than in rats that underwent only PSNL. No significant differences were observed in the right side, contralateral to PSNL. (n = 6 rats per group, *P < 0.05, ** P < 0.01, *** P < 0.001, two-way RM ANOVA followed by Bonferroni post-tests). Data are expressed as mean ± s.e.m. Arrowheads indicate day of surgery for crush injury and PSNL. Withdrawal threshold (g) Withdrawal latency (s) Withdrawal threshold (g) Withdrawal latency (s) Withdrawal threshold (g) Crush Crush Crush Crush Crush PSNL PSNL PSNL PSNL PSNL Withdrawal threshold (g) Withdrawal latency (s) Withdrawal threshold (g) Withdrawal latency (s) Withdrawal threshold (g) Crush Crush Crush Crush Crush PSNL PSNL PSNL PSNL PSNL Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 4 of 9 http://www.molecularpain.com/content/7/1/1 subpopulations of medium- to large-sized DRG neurons with about 25-30% of NF-200 neurons containing ATF+ expressing neurofilament-200 (NF-200) [16] and small- nuclei and about 28-34% of peripherin neurons contain- sized DRG neurons expressing peripherin, a marker ing ATF+ nuclei in the DRGs ipsilateral to the PSNL found predominantly in small sensory ganglion cells (left side). PSNL also induced a large increase in ED1 with unmyelinated C-fiber axons [17]. Cryosections of (Figure 2C) and T-cell receptor (Figure 2D) immunor- lumbar spinal cords were stained for microglia (ionized eactivity in the ipsilateral DRGs, compared to contralat- calcium binding adaptor molecule 1; IBA1 marker) and eral uninjured DRGs. Interestingly, there were no astrocytes (glial fibrillary acidic protein; GFAP marker). differences between preconditioned and unconditioned We found that compared to the contralateral uninjured rats in the ipsilateral DRGs; the preconditioning injury side, PSNL induced a large increase in the level of did not affect the percentage of large NF-200 positive ATF3/NF-200 (Figure 2A) and ATF3/peripherin (Figure 2B) neurons (Figure 2A) and small peripherin positive AB Injured Injured 40 40 30 30 ** *** 20 20 10 10 uninjured uninjured Ipsilateral Contralateral Ipsilateral Contralateral Injured CD Injured 1.00 10.0 0.75 7.5 0.50 5.0 uninjured uninjured 0.25 2.5 0.00 0.0 Ipsilateral Contralateral Ipsilateral Contralateral Figure 2 Effects of preconditioning nerve lesion on PSNL-induced neuronal damage and inflammation in L4/5 DRGs. (A-B) Percentage of ATF3+ DRG neurons in populations of large NF-200-expressing neurons, and small peripherin-expressing neurons. Compared to the contralateral uninjured side, a large increase in NF-200 neurons (green) containing ATF3+ nuclei (red) (A) and in peripherin positive neurons (green) containing ATF3+ nuclei (red) (B) was observed on the side ipsilateral to PSNL in all groups. On the ipsilateral side, there was no significant difference between preconditioned (crush-injured) and unconditioned (no crush) groups. On the contralateral side, the percentage of NF-200 neurons containing ATF3+ nuclei (A) and peripherin neurons containing ATF3+ nuclei (B) was significantly higher in the rats that underwent right tibial nerve crush injury as compared to all other groups. (C-D) Macrophage and T-cell presence in DRGs. Compared to the contralateral uninjured side, ED1 immunoreactivity (in green) (C) and the number of T cells (in green) (D) were markedly increased after PSNL on the ipsilateral side, but with no significant difference between preconditioned and unconditioned groups. On the contralateral side, ED1 immunoreactivity (C) and T-cell numbers (D) were significantly higher in the rats that underwent right tibial nerve crush injury as compared to all other groups. Micrographs on the right of each histogram show representative examples of immunoreactivity in DRGs from injured (ipsilateral) and uninjured (contralateral) sides. (n = 3 rats per group, *P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA followed by Bonferroni post-tests). Data are expressed as mean ± s.e.m. Scale bars represent 50 μm. ED1 Areal Fraction (%) ATF3 / NF-200 (% ) Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush ATF3 / Peripherin (% ) Number of T cells Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 5 of 9 http://www.molecularpain.com/content/7/1/1 neurons (Figure 2B) expressing ATF3, or the density of after PSNL on the ipsilateral side, and to a smaller macrophages (Figure 2C) and number of infiltrating extent on the contralateral side, as compared to normal T cells (Figure 2D) following the PSNL. On the right animals (Figure 3B). However, although both microglia side, however, crush injury of the right tibial nerve by and astrocytes were significantly upregulated (P<0.05-P itself caused a significant increase (P <0.01-P <0.001) < 0.001) in the ipsilateral dorsal and ventral horns com- in large and small ATF3+ neurons (Figure 2A, B) and a pared to normal controls, there were no significant dif- significant increase (P < 0.05) in the number of macro- ferences between the preconditioned and unconditioned phages and T cells (Figure 2C, D). groups (Figure 3A, B). In the spinal cord contralateral to In thespinalcord, aconsiderableincreaseinIBA1 the PSNL, crush injury of the right tibial nerve by itself immunoreactivity (microglia) was observed after PSNL induced a significant increase in microglia activation in on the ipsilateral side of both the dorsal and the ventral both dorsal (P < 0.001) and ventral (P < 0.05) horns horns, as compared to the contralateral uninjured side (Figure 3A), but not astrocyte activation (Figure 3B). and normal controls (Figure 3A). A large increase in Thus, a preconditioning nerve lesion prior to PSNL did GFAP immunoreactivity (astrocytes) was also observed not change the level of ligation-induced neuronal Dorsal spinal cord Ventral spinal cord Microglia Ipsilateral Contralateral Dorsal horn *** 15 * 5 *** 5 ** 0 Ventral horn Ipsilateral Contralateral Ipsilateral Contralateral B Astrocytes Ipsilateral Contralateral Dorsal horn 0 0 Ventral horn Ipsilateral Contralateral Ipsilateral Contralateral Figure 3 Effects of preconditioning nerve lesion on glial activation in lumbar spinal cord (L4-6), 1 week after partial ligation of the left sciatic nerve. (A) Activation of microglia in the ipsilateral dorsal and ventral horn of the spinal cord was significantly increased 7 days after PSNL in both preconditioned (crush-injured) and unconditioned (no crush) rats as compared to normal rats and to the contralateral uninjured side. Activation of microglia in the contralateral dorsal and ventral horn of the spinal cord was significantly increased only in the rats that underwent (2 weeks before) right tibial nerve crush injury. (B) Activation of astrocytes in the ipsilateral dorsal horn of the spinal cord was significantly increased 7 days after PSNL in both preconditioned (crush-injured) and unconditioned (no crush) groups as compared to normal rats. Astrocyte activation was significantly increased in the ventral horn of preconditioned rats (left tibial and left peroneal crush) as compared to normal rats. In the contralateral spinal cord, no significant differences were observed between the groups, except for the dorsal horn of preconditioned rats (left tibial crush) as compared to normal rats. Micrographs (right panel) show examples of immunoreactivity to IBA1 (microglia, in red) and GFAP (astrocytes, in green) in sides ipsilateral (left) and contralateral (right) to PSNL in both dorsal and ventral spinal cords of unconditioned rats. (n = 3 rats per group, *P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA followed by Bonferroni post-tests). Data are expressed as mean ± s.e.m. Scale bars represent 50 μm. GFAP Areal Fraction (%) Iba1 Areal Fraction (%) Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Normal Normal Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Normal Normal GFAP Areal Fraction (%) Iba1 Areal Fraction (%) Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Normal Normal Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Normal Normal Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 6 of 9 http://www.molecularpain.com/content/7/1/1 damage, macrophage and T-cell infiltration into DRGs, action of harmful mediators (e.g. glutamate, nitric oxide) or microglia and astrocyte activation in the spinal cord. and modifying post-injury inflammation, thereby redu- cing pain following the subsequent injury. Other possi- Discussion bilities include differential modulation of endogenous Our study shows that a preconditioning lesion distal to opioids in the spinal cord [23] of preconditioned and the experimental neuropathic injury (PSNL) inhibits the unconditioned animals, and generation of an unidenti- development of mechanical pain hypersensitivity. A left fied endogenous analgesic substance, which enters tibial nerve crush seven days before partial ligation of the blood stream and provokes a systemic protective the left sciatic nerve inhibits the development of thermal response after the preconditioning injury. Our finding of hyperalgesia and mechanical allodynia. A crush injury of differential pain behaviors following the different pre- the left peroneal nerve or the right tibial nerve only conditioning nerve lesions suggests that the nerve size, transiently attenuates mechanical allodynia, but not the type of fibers in the crushed nerve, and the extent thermal hyperalgesia induced by left PSNL. The paw of sensory afferent denervation influence the level of withdrawal response of preconditioned rats was not pain inhibition. It is likely that several mechanisms are compromised by the nerve crush injury as all rats were involved. capable of withdrawing their hindpaw in response to We found no difference in PSNL-induced neuronal mechanical or thermal stimuli. Furthermore, compared damage and macrophage and T-cell infiltration into the to sham operation, crush injury of the right tibial nerve DRGs, and no difference in PSNL-induced astrocyte and by itself (Figure 1E, right panel) induced a slight reduc- microglia activation in the spinal cords of precondi- tion in paw withdrawal threshold to mechanical stimuli tioned and unconditioned animals. Interestingly, crush during the course of the experiment. It should be noted injury of the tibial nerve by itself induced significant that combined crush injury of both the tibial and pero- neuronal damage and neuroimmune activation, and the neal nerves has previously been shown to induce subsequent PSNL did not have a substantial additive mechanical hypersensitivity of the paw [18]. effect (Figure 2, 3). Although there were no overall dif- Since the greatest effect of nerve crush on ligation- ferences in the magnitude of the neuroinflammatory induced neuropathic pain occurred following left tibial response between the study groups, there might be dif- nerve lesion, it is likely that some of the effect is due to ferences in the phenotype (e.g., cytokine profile) of the denervation of sensory afferents in the area of the mid- cells involved. Activation of spinal microglia and astro- plantar surface of the paw tested, an area corresponding cytes and concomitant release of proinflammatory to the cutaneous innervation of the tibial nerve [18]. products are strongly implicated in pathological neuro- However, a recent study has demonstrated that even pathic pain [24-26]. Microglia cells, in particular, are after total tibial nerve axotomy, some evoked pain beha- emerging as critical players in peripheral injury-induced viors are observed in the tibial-innervated skin [19]. In neuropathic pain [27]. In contrast, our data suggest that addition, crush injury of the common peroneal nerve the general level of spinal glia activation is not necessa- predominantly innervating the lateral half of the hairy rily correlated with the level of painsensitivity.Insup- skin of the paw [20] and crush injury of the right tibial port of this, previous studies have demonstrated that nerve innervating the right hindpaw, also had an effect neuropathic pain behaviors preceded and did not strictly in our study. These results indicate the involvement of correlate with microglial responses following peripheral either systemic or central mechanisms in the precondi- nerve injury [28]. Additionally, two different models of tioning-induced pain inhibition. cancer pain exhibiting pain hypersensitivity resulted in The mechanisms responsible for the behavioral effects severe spinal astrogliosis without activation of microglia of preconditioning on neuropathic pain (Figure 1) are [29]. It has been suggested that microglia do not com- currently unclear. Potential mechanisms include modu- prise a single, uniform cell population, but rather cells lation of endogenous pain controls following the initial with diverse phenotypes and that microglial commit- injury, such as induction of DNIC involving inhibition ment to a phenotype can be changed depending on the of dorsal nociceptive neurons [12] or enhancement of nature of stimuli [30]. Thus, there might be a distinct descending net inhibition from supraspinal structures as pattern of glial activation that confers a beneficial neu- seen following peripheral inflammation [21]. The pre- roprotective effect as opposed to a detrimental excito- conditioning injury may trigger adaptive responses in toxic effect [30] following peripheral nerve injury resident cells within the nervous system by inducing preceded by preconditioning. Indeed, glial activation in changes in gene expression and/or post-translational the nervous system has been shown in some instances modifications of existing proteins [22]. Some of these to be neuroprotective by release of anti-inflammatory changes may serve to protect the tissue by stabilizing factors and by protection against neuronal damage cell energy and protein metabolism, ameliorating the [24,31]. Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 7 of 9 http://www.molecularpain.com/content/7/1/1 animal, in which the sciatic nerve was exposed but not Conclusions damaged in any way. Muscle layers were closed with 4-0 In summary, our results show that pain sensation might silk sutures and the skin wounds closed with skin be affected by a prior injury. The mechanisms underly- staples. ing the conditioning-induced attenuation of pain hyper- sensitivity following neuropathic injury are not known. Behavioral testing It is logical to assume that the long-lasting protective Rats were habituated to the behavioral testing apparatus effect by the preconditioning tibial nerve crush injury is for 30 min to 1 hr prior to data collection and the test- at least partially due to denervation of the skin exam- ined by the sensory tests employed in the present study. ing environment was kept quiet and well controlled. Behavioral tests were performed 3 times a week for 30 However, the transient inhibition of mechanical allody- days, with a baseline measurement before surgery. Ther- nia by the preconditioning crush injury of the peroneal mal hyperalgesia was assessed as previously described nerve, and the tibial nerve on the other side of the [33], by exposing the mid-plantar surface of the hindpaw PSNL, suggests an involvement of systemic and/or cen- to a beam of radiant heat through a transparent glass tral changes in the preconditioned animals. Identifying surface using a plantar analgesia meter for paw stimula- the underlying mechanisms may have important impli- tion (Ugo Basile, Italy). The latency of withdrawal from cations for the understanding of persistent pain that the heat stimulus was automatically recorded as the develops after nerve injury and for developing treatment time taken from the onset of radiant heat stimulation to approaches to ease neuropathic pain. withdrawal of the paw. A cut-off latency of 22 sec was pre-set to prevent tissue damage. Mechanical allodynia Methods was assessed by placing an animal on an elevated wire Animals grid and stimulating the plantar surface of the hindpaw, Inbred male Wistar rats (Biological Resources Centre, using an electronic von Frey anesthesiometer (IITC Inc., University of New South Wales, Australia), 7-8 weeks of Woodland Hills, CA, USA). The probe was gently age at the commencement of study were used. Animals applied to the centre of the paw just posterior to the were housed at approximately 22°C in groups of six paw pads with slowly increasing force until the rat with- under a 12-h light/dark cycle with free access to food drew its paw in response to the stimulus. The device and water. Protocols were approved by the Animal Care automatically recorded and displayed the force (in and Ethics Committee of the University of New South grams) that elicited a withdrawal response. Thermal Wales and adhered to the guidelines of the Committee latencies and mechanical thresholds were measured four for Research and Ethical Issues of the International Association for the Study of Pain. times for each paw, with a 3-5 min interval between measurements and the mean was calculated. All beha- vioral experiments were repeated twice by different Nerve crush injury experimenters. Animals were anesthetized with halothane in a 1:1 mix- ture of O and N O and the sciatic nerve was exposed 2 2 Immunohistochemistry distally with its three terminal branches: the sural, com- Seven days after partial ligation of the sciatic nerve, rats mon peroneal and tibial nerves. The tibial or peroneal were anesthetized using an overdose of sodium pento- nervewas crushedfor 30 secondsbyapairoffinefor- barbitone (120 mg/kg i.p.). They were then perfused ceps with a smooth flat crushing surface. The crush through the aorta with 0.9% saline containing heparin injury resulted in a flattened and transparent section of followed by fresh 4% paraformaldehyde in 0.1 M phos- nerve at the crush area. Sham controls involved expo- phate buffer (pH 7.4) for tissue fixation. Both left and sure of the relevant nerve without any lesion. Muscle right L4 and L5 DRGs as well as L4-L6 lumbar spinal was closed in layers by suturing and the skin clipped. cord segments were harvested. Tissues were post-fixed Partial ligation of the sciatic nerve in 4% paraformaldehyde for 6 h and then transferred to The surgical procedure was based on that described by 30% sucrose overnight. Cryosections (10-20 μmthick) Seltzer et al [32]. Rats were anesthetized with halothane were prepared and stained as previously described [34]. in a1:1 mixtureofO and N O. An incision was made DRG sections were stained for T cells with mouse anti- 2 2 at the proximal thigh and the left sciatic nerve exposed. rat monoclonal antibody to ab T-cell receptor, clone About one third of the diameter of the left sciatic nerve R73 (1:200; BD Biosciences-PharMingen, San Diego, CA, was tightly ligated just proximal to its branch to the USA) and for macrophages with mouse anti-rat CD68, posterior biceps and semitendinosus muscles, using 7-0 clone ED1 (1:250; Serotec, Oxford, UK). Double labeling silk (Tyco Healthcare, Norwalk, CT, USA). A sham was performed with rabbit anti-ATF3 (1:400; Santa Cruz operation was carried out on the right hind limb of each Biotechnology, Santa Cruz, CA, USA) and either mouse Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 8 of 9 http://www.molecularpain.com/content/7/1/1 NF-200: neurofilament-200; IBA1: ionized calcium binding adaptor molecule anti-NF-200 (1:500; Sigma, Castle Hill, New South 1; GFAP: glial fibrillary acidic protein; ANOVA: analysis of variance. Wales, Australia) or mouse anti-peripherin (1:400; Che- micon International, Billerica, MA, USA). Spinal cord Acknowledgements This work was supported by grants to GMT from the Faculty of Medicine, sections were stained for microglia with rabbit anti- The University of New South Wales, the National Health and Medical IBA1 (1:2000; Wako, Osaka, Japan) and for astrocytes Research Council of Australia (ID # 568637), and the NSW Office for Science with mouse anti-GFAP (1:2000; Chemicon Interna- and Medical Research. tional). Sections were blocked and then incubated with Author details the primary antibody. Elimination of the primary anti- School of Medical Sciences, University of New South Wales, Sydney, NSW body wasusedasanegative control. Thesections were 2052 Australia. Department of Neurobiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China. washed 4 times and incubated with a secondary anti- Department of Physiology, University of Sydney, NSW 2006 Australia. body as appropriate: donkey anti-mouse IgG conjugated with Cy2 (1:100; Jackson ImmunoResearch, West Grove, Authors’ contributions GM-T designed and coordinated the study, carried out some experiments, PA, USA) or donkey anti-rabbit IgG conjugated with analyzed data and wrote the manuscript. ML and HNA carried out some Cy3 (1:400; Jackson ImmunoResearch). In the case of behavioral testing, tissue processing and immunohistochemical analysis. AW double labeling, both secondaryantibodieswereused. and DJT participated in study design and data analysis. All authors read and approved the final manuscript. Sections were washed 4 times and treated with fluores- cent mounting medium (DakoCytomation) before being Competing interests cover-slipped. The authors declare that they have no competing interests. Received: 22 February 2010 Accepted: 5 January 2011 Image analysis Published: 5 January 2011 Sections were viewed on an Olympus fluorescence microscope. Images were captured using an Olympus References 1. Moalem G, Tracey DJ: Immune and inflammatory mechanisms in DP70 camera and DP Controller software (Olympus neuropathic pain. Brain Res Rev 2006, 51:240-264. Tokyo, Japan) and were taken from 3-6 sections in each 2. McQuarrie IG, Grafstein B: Axon outgrowth enhanced by a previous nerve animal. In each photograph, immunoreactivity was eval- injury. Archives of Neurology 1973, 29:53-55. 3. 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Submit your next manuscript to BioMed Central doi:10.1186/1744-8069-7-1 and take full advantage of: Cite this article as: Moalem-Taylor et al.: A preconditioning nerve lesion inhibits mechanical pain hypersensitivity following subsequent neuropathic injury. Molecular Pain 2011 7:1. • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Pain Springer Journals

A preconditioning nerve lesion inhibits mechanical pain hypersensitivity following subsequent neuropathic injury

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Copyright © 2011 by Moalem-Taylor et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology
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Abstract

Background: A preconditioning stimulus can trigger a neuroprotective phenotype in the nervous system - a preconditioning nerve lesion causes a significant increase in axonal regeneration, and cerebral preconditioning protects against subsequent ischemia. We hypothesized that a preconditioning nerve lesion induces gene/protein modifications, neuronal changes, and immune activation that may affect pain sensation following subsequent nerve injury. We examined whether a preconditioning lesion affects neuropathic pain and neuroinflammation after peripheral nerve injury. Results: We found that a preconditioning crush injury to a terminal branch of the sciatic nerve seven days before partial ligation of the sciatic nerve (PSNL; a model of neuropathic pain) induced a significant attenuation of pain hypersensitivity, particularly mechanical allodynia. A preconditioning lesion of the tibial nerve induced a long-term significant increase in paw-withdrawal threshold to mechanical stimuli and paw-withdrawal latency to thermal stimuli, after PSNL. A preconditioning lesion of the common peroneal induced a smaller but significant short-term increase in paw-withdrawal threshold to mechanical stimuli, after PSNL. There was no difference between preconditioned and unconditioned animals in neuronal damage and macrophage and T-cell infiltration into the dorsal root ganglia (DRGs) or in astrocyte and microglia activation in the spinal dorsal and ventral horns. Conclusions: These results suggest that prior exposure to a mild nerve lesion protects against adverse effects of subsequent neuropathic injury, and that this conditioning-induced inhibition of pain hypersensitivity is not dependent on neuroinflammation in DRGs and spinal cord. Identifying the underlying mechanisms may have important implications for the understanding of neuropathic pain due to nerve injury. Background (a branch of the sciatic nerve), made 2 weeks before Peripheral nerve injury often results in neuropathic pain sciatic nerve injury, causes a significant increase in axon characterized by unpleasant and persistent increases in outgrowth [2,3]. Further work has shown that regenera- pain sensitivity, including hyperalgesia and allodynia. It tion of dorsal root ganglion (DRG) central processes is well recognized that nerve lesion induces neuronal into a peripheral nerve graft and regeneration of dorsal and immunological changes and modifies gene and pro- column fibers into a spinal cord lesion site is signifi- tein expression, both in the peripheral nervous system cantly improved by performing a peripheral nerve lesion and in the spinal cord [1]. However, whether such [4-6]. Thus, a preconditioning nerve lesion appears to changes affect neuropathic pain behavior following sub- increase the intrinsic regenerative ability of central sequent injury is not known. axons by inducing molecular changes (e.g. elevation of Studies on axonal regeneration in the sciatic nerve intracellular cyclic AMP) that allow axons to overcome have shown that a conditioning lesion of the tibial nerve myelin inhibition [7]. Studies on cerebral precondition- ing have shown that a brief period of sub-lethal or mild preconditioning ischemia attenuates injury from subse- * Correspondence: gila@unsw.edu.au School of Medical Sciences, University of New South Wales, Sydney, NSW quent severe ischemia [8]. This neuroprotection is 2052 Australia achieved by promoting neuronal survival through Full list of author information is available at the end of the article © 2011 Moalem-Taylor et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 2 of 9 http://www.molecularpain.com/content/7/1/1 attenuation of several injury-inducing mechanisms (e.g. with either the contralateral side or baseline values excitotoxicity, ion/pH imbalance, oxidative stress, meta- before surgery, as indicated by a sharp decrease in paw bolic dysfunction, inflammation) and through enhance- withdrawal threshold to mechanical stimuli and paw ment of endogenous repair processes [9]. withdrawal latency to thermal stimuli (Figure 1). On the There are limited and conflicting data regarding the contralateral side to PSNL (right hindpaws), there were effects of an existing injury on the development of pain no significant differences in either the mechanical or the subsequent to a second injury. Some studies have thermal pain sensitivity between the preconditioned demonstrated that noxious cutaneous stimuli elicit a (crush-injured) and unconditioned (no crush) rats powerful and long-lasting inhibition of spinal dorsal (Figure 1 right panel). However, on the ipsilateral side horn and trigeminal convergent neurons, termed diffuse to PSNL (left hindpaws), a preconditioning lesion of the noxious inhibitory controls (DNIC) [10,11]. DNIC left tibial nerve induced a long-term significant attenua- effects are directly related to the duration of the condi- tion of both ligation-induced mechanical allodynia tioning painful stimulus [11] and predict inhibition of (Figure 1A) and thermal hyperalgesia (Figure 1B). Com- pain behaviors following prior painful insults. Indeed, pared with unconditioned rats, preconditioned rats had activation of DNIC was confirmed in rats with chronic significantly higher (1.6-2.5-fold; P < 0.01-P < 0.001) constriction injury of the sciatic nerve, demonstrating paw withdrawal thresholds to mechanical stimuli 2-19 increased inhibition by nociceptive conditioning stimuli days post-PSNL (Figure 1A) and significantly higher to the hindpaw [12]. In contrast, other studies have (1.5-1.6-fold; P <0.05-P < 0.001) paw withdrawal laten- shown that a trigeminal nerve injury significantly accel- cies to thermal stimuli 2-9 days post-PSNL (Figure 1B). erated the development of mechanical allodynia and A preconditioning lesion of the left common peroneal hyperalgesia following a chronic constriction injury of induced a smaller but significant short-term attenuation the sciatic nerve as a second injury (day 7), in Lewis but of mechanical allodynia (Figure 1C), but not thermal not Sprague-Dawley or Sabra rats [13]. Repeated injury hyperalgesia (Figure 1D). Compared with unconditioned to the lumbar nerve roots at 42 days also produced rats, preconditioned (left peroneal) rats had significantly enhanced mechanical allodynia and spinal neuroinflam- higher (1.6-1.9-fold; P < 0.05-P < 0.01) paw withdrawal mation [14]. Thus, it appears that modulation of pain thresholds to mechanical stimuli 7-12 days post-PSNL following a primary injury is greatly dependent on the (Figure 1C). These preconditioned (left peroneal) rats location and timing of injuries, the nature of stimulus, also had higher (1.2-1.4-fold) paw withdrawal latencies and the strain of animals. to thermal stimuli 7-9 days post-PSNL (Figure 1D) com- Here, using a rat model of neuropathic pain we inves- pared with unconditioned rats, but this effect was not tigated whether a preconditioning nerve lesion influ- statistically significant. A preconditioning lesion of the ences pain sensation and neuroinflammation following a right tibial nerve induced a small decrease in paw with- subsequent distant peripheral nerve injury involving the drawal thresholds to mechanical stimuli in the contralat- same dorsal root ganglia. eral side to PSNL, though these changes were not significant (Figure 1E, right). On the ipsilateral side, a Results short-term attenuation of mechanical allodynia was A preconditioning nerve lesion alters pain behaviors observed, and preconditioned (right tibial) rats had a following neuropathic injury significantly higher (1.6-fold; P <0.05) pawwithdrawal To study the effects of preconditioning on neuropathic threshold to mechanical stimuli 4 days post-PSNL (Fig- pain, we carried out a crush injury to one of the term- ure 1E). There were no differences in thermal pain sen- inal branches of the sciatic nerve or exposed the nerve sitivity between unconditioned and preconditioned branch without crush injury (sham control), 1 week (right tibial) rats on either the ipsilateral or the contral- before partial ligation of the sciatic nerve (PSNL; a ateral sides to PSNL (data not shown). model of neuropathic pain) and measured pain beha- viors for 30 days. The following groups of rats (n = 6 A preconditioning nerve lesion does not affect PSNL- animals per group) were used: (1) Left tibial nerve crush induced neuroinflammation in DRG and spinal cord injury 1 week before left PSNL; (2) Left common pero- We next examined whether a preconditioning nerve neal nerve crush injury 1 week before left PSNL; (3) lesion affects neuroinflammation 7 days after PSNL (14 Right tibial nerve crush injury 1 week before left PSNL. days after crush injury) in L4/5 DRGs and in L4-6 spinal Each of these preconditioned groups was compared to a cord segments using immunohistochemistry (n = 3 ani- control group with a relevant sham crush 1 week before mals per group). Cryosections of excised DRGs were left PSNL (unconditioned). stained for macrophages (ED1 marker) and T cells (T- Following PSNL, all unconditioned rats developed pain cell receptor marker) [15], and for damaged neurons hypersensitivity in the paw ipsilateral to PSNL compared expressing activating transcription factor 3 (ATF3) in Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 3 of 9 http://www.molecularpain.com/content/7/1/1 Ipsilateral Contralateral 30 30 Left tibial crush+PSNL Sham+PSNL 25 25 20 20 15 15 *** ** Left tibial crush+PSNL 10 *** 10 *** *** Sham+PSNL ** *** 5 5 *** 0 0 -5 -2 1 4 7 10 13 16 19 22 25 28 31 -5 -2 1 4 7 10 13 16 19 22 25 28 31 Days Days 20 20 Left tibial crush+PSNL 18 18 Sham+PSNL 16 16 14 ** 14 12 12 10 10 8 8 6 6 ** *** ** 4 4 Left tibial crush+PSNL 2 2 Sham+PSNL 0 0 -5 -2 1 4 7 10 13 16 19 22 25 28 31 -5 -2 1 4 7 10 13 16 19 22 25 28 31 Days Days 35 35 Left peroneal crush+PSNL 30 30 Sham+PSNL 25 25 20 20 15 15 10 10 Left peroneal crush+PSNL ** 5 5 Sham+PSNL 0 0 -2 1 4 7 10 13 16 19 22 25 28 31 -2 1 4 7 10 13 16 19 22 25 28 31 Days Days 20 20 18 18 Left peroneal crush+PSNL 16 16 Sham+PSNL 14 14 12 12 10 10 8 8 6 6 4 4 Left peroneal crush+PSNL 2 2 Sham+PSNL 0 0 -2 1 4 7 10 13 16 19 22 25 28 31 -2 1 4 7 10 13 16 19 22 25 28 31 Days Days E 30 30 Right tibial crush+PSNL 25 25 Sham+PSNL 20 20 15 15 10 10 * Right tibial crush+PSNL 5 5 Sham+PSNL 0 0 -5 -2 1 4 7 10 13 16 19 22 25 28 31 -5 -2 1 4 7 10 13 16 19 22 25 28 31 Days Days Figure 1 Effects of preconditioning nerve lesion on neuropathic pain behaviors due to partial ligation of the left sciatic nerve. (A-B) Tibial nerve crush injury (left) inhibits PSNL-induced mechanical and thermal pain hypersensitivity. Withdrawal thresholds to mechanical stimuli (d9-d26) (A) and withdrawal latencies to thermal stimuli (d9-d16) (B) were significantly greater in the ipsilateral left hindpaws of rats that underwent left tibial crush injury 1 week before PSNL than in rats that underwent only PSNL. No significant differences in paw withdrawal thresholds (A) and in paw withdrawal latencies (B) were observed in the contralateral right side. (C-D) Peroneal nerve crush injury (left) inhibits PSNL-induced mechanical, but not thermal, pain hypersensitivity. Withdrawal thresholds to mechanical stimuli (d14-d19) (C) were significantly greater in ipsilateral left hindpaws of rats that underwent left peroneal crush injury 1 week before PSNL than in rats that underwent only PSNL. No significant differences in paw withdrawal thresholds were observed in the contralateral right side. (D) No significant differences were observed in paw withdrawal latencies to thermal stimuli in both the ipsilateral and contralateral sides. (E) Tibial nerve crush injury (right; on the other side of the PSNL) transiently inhibits PSNL-induced mechanical pain hypersensitivity. Withdrawal thresholds to mechanical stimuli (d11) were significantly greater in ipsilateral left hindpaws of rats that underwent right tibial crush injury 1 week before PSNL than in rats that underwent only PSNL. No significant differences were observed in the right side, contralateral to PSNL. (n = 6 rats per group, *P < 0.05, ** P < 0.01, *** P < 0.001, two-way RM ANOVA followed by Bonferroni post-tests). Data are expressed as mean ± s.e.m. Arrowheads indicate day of surgery for crush injury and PSNL. Withdrawal threshold (g) Withdrawal latency (s) Withdrawal threshold (g) Withdrawal latency (s) Withdrawal threshold (g) Crush Crush Crush Crush Crush PSNL PSNL PSNL PSNL PSNL Withdrawal threshold (g) Withdrawal latency (s) Withdrawal threshold (g) Withdrawal latency (s) Withdrawal threshold (g) Crush Crush Crush Crush Crush PSNL PSNL PSNL PSNL PSNL Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 4 of 9 http://www.molecularpain.com/content/7/1/1 subpopulations of medium- to large-sized DRG neurons with about 25-30% of NF-200 neurons containing ATF+ expressing neurofilament-200 (NF-200) [16] and small- nuclei and about 28-34% of peripherin neurons contain- sized DRG neurons expressing peripherin, a marker ing ATF+ nuclei in the DRGs ipsilateral to the PSNL found predominantly in small sensory ganglion cells (left side). PSNL also induced a large increase in ED1 with unmyelinated C-fiber axons [17]. Cryosections of (Figure 2C) and T-cell receptor (Figure 2D) immunor- lumbar spinal cords were stained for microglia (ionized eactivity in the ipsilateral DRGs, compared to contralat- calcium binding adaptor molecule 1; IBA1 marker) and eral uninjured DRGs. Interestingly, there were no astrocytes (glial fibrillary acidic protein; GFAP marker). differences between preconditioned and unconditioned We found that compared to the contralateral uninjured rats in the ipsilateral DRGs; the preconditioning injury side, PSNL induced a large increase in the level of did not affect the percentage of large NF-200 positive ATF3/NF-200 (Figure 2A) and ATF3/peripherin (Figure 2B) neurons (Figure 2A) and small peripherin positive AB Injured Injured 40 40 30 30 ** *** 20 20 10 10 uninjured uninjured Ipsilateral Contralateral Ipsilateral Contralateral Injured CD Injured 1.00 10.0 0.75 7.5 0.50 5.0 uninjured uninjured 0.25 2.5 0.00 0.0 Ipsilateral Contralateral Ipsilateral Contralateral Figure 2 Effects of preconditioning nerve lesion on PSNL-induced neuronal damage and inflammation in L4/5 DRGs. (A-B) Percentage of ATF3+ DRG neurons in populations of large NF-200-expressing neurons, and small peripherin-expressing neurons. Compared to the contralateral uninjured side, a large increase in NF-200 neurons (green) containing ATF3+ nuclei (red) (A) and in peripherin positive neurons (green) containing ATF3+ nuclei (red) (B) was observed on the side ipsilateral to PSNL in all groups. On the ipsilateral side, there was no significant difference between preconditioned (crush-injured) and unconditioned (no crush) groups. On the contralateral side, the percentage of NF-200 neurons containing ATF3+ nuclei (A) and peripherin neurons containing ATF3+ nuclei (B) was significantly higher in the rats that underwent right tibial nerve crush injury as compared to all other groups. (C-D) Macrophage and T-cell presence in DRGs. Compared to the contralateral uninjured side, ED1 immunoreactivity (in green) (C) and the number of T cells (in green) (D) were markedly increased after PSNL on the ipsilateral side, but with no significant difference between preconditioned and unconditioned groups. On the contralateral side, ED1 immunoreactivity (C) and T-cell numbers (D) were significantly higher in the rats that underwent right tibial nerve crush injury as compared to all other groups. Micrographs on the right of each histogram show representative examples of immunoreactivity in DRGs from injured (ipsilateral) and uninjured (contralateral) sides. (n = 3 rats per group, *P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA followed by Bonferroni post-tests). Data are expressed as mean ± s.e.m. Scale bars represent 50 μm. ED1 Areal Fraction (%) ATF3 / NF-200 (% ) Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush ATF3 / Peripherin (% ) Number of T cells Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 5 of 9 http://www.molecularpain.com/content/7/1/1 neurons (Figure 2B) expressing ATF3, or the density of after PSNL on the ipsilateral side, and to a smaller macrophages (Figure 2C) and number of infiltrating extent on the contralateral side, as compared to normal T cells (Figure 2D) following the PSNL. On the right animals (Figure 3B). However, although both microglia side, however, crush injury of the right tibial nerve by and astrocytes were significantly upregulated (P<0.05-P itself caused a significant increase (P <0.01-P <0.001) < 0.001) in the ipsilateral dorsal and ventral horns com- in large and small ATF3+ neurons (Figure 2A, B) and a pared to normal controls, there were no significant dif- significant increase (P < 0.05) in the number of macro- ferences between the preconditioned and unconditioned phages and T cells (Figure 2C, D). groups (Figure 3A, B). In the spinal cord contralateral to In thespinalcord, aconsiderableincreaseinIBA1 the PSNL, crush injury of the right tibial nerve by itself immunoreactivity (microglia) was observed after PSNL induced a significant increase in microglia activation in on the ipsilateral side of both the dorsal and the ventral both dorsal (P < 0.001) and ventral (P < 0.05) horns horns, as compared to the contralateral uninjured side (Figure 3A), but not astrocyte activation (Figure 3B). and normal controls (Figure 3A). A large increase in Thus, a preconditioning nerve lesion prior to PSNL did GFAP immunoreactivity (astrocytes) was also observed not change the level of ligation-induced neuronal Dorsal spinal cord Ventral spinal cord Microglia Ipsilateral Contralateral Dorsal horn *** 15 * 5 *** 5 ** 0 Ventral horn Ipsilateral Contralateral Ipsilateral Contralateral B Astrocytes Ipsilateral Contralateral Dorsal horn 0 0 Ventral horn Ipsilateral Contralateral Ipsilateral Contralateral Figure 3 Effects of preconditioning nerve lesion on glial activation in lumbar spinal cord (L4-6), 1 week after partial ligation of the left sciatic nerve. (A) Activation of microglia in the ipsilateral dorsal and ventral horn of the spinal cord was significantly increased 7 days after PSNL in both preconditioned (crush-injured) and unconditioned (no crush) rats as compared to normal rats and to the contralateral uninjured side. Activation of microglia in the contralateral dorsal and ventral horn of the spinal cord was significantly increased only in the rats that underwent (2 weeks before) right tibial nerve crush injury. (B) Activation of astrocytes in the ipsilateral dorsal horn of the spinal cord was significantly increased 7 days after PSNL in both preconditioned (crush-injured) and unconditioned (no crush) groups as compared to normal rats. Astrocyte activation was significantly increased in the ventral horn of preconditioned rats (left tibial and left peroneal crush) as compared to normal rats. In the contralateral spinal cord, no significant differences were observed between the groups, except for the dorsal horn of preconditioned rats (left tibial crush) as compared to normal rats. Micrographs (right panel) show examples of immunoreactivity to IBA1 (microglia, in red) and GFAP (astrocytes, in green) in sides ipsilateral (left) and contralateral (right) to PSNL in both dorsal and ventral spinal cords of unconditioned rats. (n = 3 rats per group, *P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA followed by Bonferroni post-tests). Data are expressed as mean ± s.e.m. Scale bars represent 50 μm. GFAP Areal Fraction (%) Iba1 Areal Fraction (%) Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Normal Normal Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Normal Normal GFAP Areal Fraction (%) Iba1 Areal Fraction (%) Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Normal Normal Left tibial crush Left tibial crush Left peroneal crush Left peroneal crush No crush No crush Right tibial crush Right tibial crush Normal Normal Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 6 of 9 http://www.molecularpain.com/content/7/1/1 damage, macrophage and T-cell infiltration into DRGs, action of harmful mediators (e.g. glutamate, nitric oxide) or microglia and astrocyte activation in the spinal cord. and modifying post-injury inflammation, thereby redu- cing pain following the subsequent injury. Other possi- Discussion bilities include differential modulation of endogenous Our study shows that a preconditioning lesion distal to opioids in the spinal cord [23] of preconditioned and the experimental neuropathic injury (PSNL) inhibits the unconditioned animals, and generation of an unidenti- development of mechanical pain hypersensitivity. A left fied endogenous analgesic substance, which enters tibial nerve crush seven days before partial ligation of the blood stream and provokes a systemic protective the left sciatic nerve inhibits the development of thermal response after the preconditioning injury. Our finding of hyperalgesia and mechanical allodynia. A crush injury of differential pain behaviors following the different pre- the left peroneal nerve or the right tibial nerve only conditioning nerve lesions suggests that the nerve size, transiently attenuates mechanical allodynia, but not the type of fibers in the crushed nerve, and the extent thermal hyperalgesia induced by left PSNL. The paw of sensory afferent denervation influence the level of withdrawal response of preconditioned rats was not pain inhibition. It is likely that several mechanisms are compromised by the nerve crush injury as all rats were involved. capable of withdrawing their hindpaw in response to We found no difference in PSNL-induced neuronal mechanical or thermal stimuli. Furthermore, compared damage and macrophage and T-cell infiltration into the to sham operation, crush injury of the right tibial nerve DRGs, and no difference in PSNL-induced astrocyte and by itself (Figure 1E, right panel) induced a slight reduc- microglia activation in the spinal cords of precondi- tion in paw withdrawal threshold to mechanical stimuli tioned and unconditioned animals. Interestingly, crush during the course of the experiment. It should be noted injury of the tibial nerve by itself induced significant that combined crush injury of both the tibial and pero- neuronal damage and neuroimmune activation, and the neal nerves has previously been shown to induce subsequent PSNL did not have a substantial additive mechanical hypersensitivity of the paw [18]. effect (Figure 2, 3). Although there were no overall dif- Since the greatest effect of nerve crush on ligation- ferences in the magnitude of the neuroinflammatory induced neuropathic pain occurred following left tibial response between the study groups, there might be dif- nerve lesion, it is likely that some of the effect is due to ferences in the phenotype (e.g., cytokine profile) of the denervation of sensory afferents in the area of the mid- cells involved. Activation of spinal microglia and astro- plantar surface of the paw tested, an area corresponding cytes and concomitant release of proinflammatory to the cutaneous innervation of the tibial nerve [18]. products are strongly implicated in pathological neuro- However, a recent study has demonstrated that even pathic pain [24-26]. Microglia cells, in particular, are after total tibial nerve axotomy, some evoked pain beha- emerging as critical players in peripheral injury-induced viors are observed in the tibial-innervated skin [19]. In neuropathic pain [27]. In contrast, our data suggest that addition, crush injury of the common peroneal nerve the general level of spinal glia activation is not necessa- predominantly innervating the lateral half of the hairy rily correlated with the level of painsensitivity.Insup- skin of the paw [20] and crush injury of the right tibial port of this, previous studies have demonstrated that nerve innervating the right hindpaw, also had an effect neuropathic pain behaviors preceded and did not strictly in our study. These results indicate the involvement of correlate with microglial responses following peripheral either systemic or central mechanisms in the precondi- nerve injury [28]. Additionally, two different models of tioning-induced pain inhibition. cancer pain exhibiting pain hypersensitivity resulted in The mechanisms responsible for the behavioral effects severe spinal astrogliosis without activation of microglia of preconditioning on neuropathic pain (Figure 1) are [29]. It has been suggested that microglia do not com- currently unclear. Potential mechanisms include modu- prise a single, uniform cell population, but rather cells lation of endogenous pain controls following the initial with diverse phenotypes and that microglial commit- injury, such as induction of DNIC involving inhibition ment to a phenotype can be changed depending on the of dorsal nociceptive neurons [12] or enhancement of nature of stimuli [30]. Thus, there might be a distinct descending net inhibition from supraspinal structures as pattern of glial activation that confers a beneficial neu- seen following peripheral inflammation [21]. The pre- roprotective effect as opposed to a detrimental excito- conditioning injury may trigger adaptive responses in toxic effect [30] following peripheral nerve injury resident cells within the nervous system by inducing preceded by preconditioning. Indeed, glial activation in changes in gene expression and/or post-translational the nervous system has been shown in some instances modifications of existing proteins [22]. Some of these to be neuroprotective by release of anti-inflammatory changes may serve to protect the tissue by stabilizing factors and by protection against neuronal damage cell energy and protein metabolism, ameliorating the [24,31]. Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 7 of 9 http://www.molecularpain.com/content/7/1/1 animal, in which the sciatic nerve was exposed but not Conclusions damaged in any way. Muscle layers were closed with 4-0 In summary, our results show that pain sensation might silk sutures and the skin wounds closed with skin be affected by a prior injury. The mechanisms underly- staples. ing the conditioning-induced attenuation of pain hyper- sensitivity following neuropathic injury are not known. Behavioral testing It is logical to assume that the long-lasting protective Rats were habituated to the behavioral testing apparatus effect by the preconditioning tibial nerve crush injury is for 30 min to 1 hr prior to data collection and the test- at least partially due to denervation of the skin exam- ined by the sensory tests employed in the present study. ing environment was kept quiet and well controlled. Behavioral tests were performed 3 times a week for 30 However, the transient inhibition of mechanical allody- days, with a baseline measurement before surgery. Ther- nia by the preconditioning crush injury of the peroneal mal hyperalgesia was assessed as previously described nerve, and the tibial nerve on the other side of the [33], by exposing the mid-plantar surface of the hindpaw PSNL, suggests an involvement of systemic and/or cen- to a beam of radiant heat through a transparent glass tral changes in the preconditioned animals. Identifying surface using a plantar analgesia meter for paw stimula- the underlying mechanisms may have important impli- tion (Ugo Basile, Italy). The latency of withdrawal from cations for the understanding of persistent pain that the heat stimulus was automatically recorded as the develops after nerve injury and for developing treatment time taken from the onset of radiant heat stimulation to approaches to ease neuropathic pain. withdrawal of the paw. A cut-off latency of 22 sec was pre-set to prevent tissue damage. Mechanical allodynia Methods was assessed by placing an animal on an elevated wire Animals grid and stimulating the plantar surface of the hindpaw, Inbred male Wistar rats (Biological Resources Centre, using an electronic von Frey anesthesiometer (IITC Inc., University of New South Wales, Australia), 7-8 weeks of Woodland Hills, CA, USA). The probe was gently age at the commencement of study were used. Animals applied to the centre of the paw just posterior to the were housed at approximately 22°C in groups of six paw pads with slowly increasing force until the rat with- under a 12-h light/dark cycle with free access to food drew its paw in response to the stimulus. The device and water. Protocols were approved by the Animal Care automatically recorded and displayed the force (in and Ethics Committee of the University of New South grams) that elicited a withdrawal response. Thermal Wales and adhered to the guidelines of the Committee latencies and mechanical thresholds were measured four for Research and Ethical Issues of the International Association for the Study of Pain. times for each paw, with a 3-5 min interval between measurements and the mean was calculated. All beha- vioral experiments were repeated twice by different Nerve crush injury experimenters. Animals were anesthetized with halothane in a 1:1 mix- ture of O and N O and the sciatic nerve was exposed 2 2 Immunohistochemistry distally with its three terminal branches: the sural, com- Seven days after partial ligation of the sciatic nerve, rats mon peroneal and tibial nerves. The tibial or peroneal were anesthetized using an overdose of sodium pento- nervewas crushedfor 30 secondsbyapairoffinefor- barbitone (120 mg/kg i.p.). They were then perfused ceps with a smooth flat crushing surface. The crush through the aorta with 0.9% saline containing heparin injury resulted in a flattened and transparent section of followed by fresh 4% paraformaldehyde in 0.1 M phos- nerve at the crush area. Sham controls involved expo- phate buffer (pH 7.4) for tissue fixation. Both left and sure of the relevant nerve without any lesion. Muscle right L4 and L5 DRGs as well as L4-L6 lumbar spinal was closed in layers by suturing and the skin clipped. cord segments were harvested. Tissues were post-fixed Partial ligation of the sciatic nerve in 4% paraformaldehyde for 6 h and then transferred to The surgical procedure was based on that described by 30% sucrose overnight. Cryosections (10-20 μmthick) Seltzer et al [32]. Rats were anesthetized with halothane were prepared and stained as previously described [34]. in a1:1 mixtureofO and N O. An incision was made DRG sections were stained for T cells with mouse anti- 2 2 at the proximal thigh and the left sciatic nerve exposed. rat monoclonal antibody to ab T-cell receptor, clone About one third of the diameter of the left sciatic nerve R73 (1:200; BD Biosciences-PharMingen, San Diego, CA, was tightly ligated just proximal to its branch to the USA) and for macrophages with mouse anti-rat CD68, posterior biceps and semitendinosus muscles, using 7-0 clone ED1 (1:250; Serotec, Oxford, UK). Double labeling silk (Tyco Healthcare, Norwalk, CT, USA). A sham was performed with rabbit anti-ATF3 (1:400; Santa Cruz operation was carried out on the right hind limb of each Biotechnology, Santa Cruz, CA, USA) and either mouse Moalem-Taylor et al. Molecular Pain 2011, 7:1 Page 8 of 9 http://www.molecularpain.com/content/7/1/1 NF-200: neurofilament-200; IBA1: ionized calcium binding adaptor molecule anti-NF-200 (1:500; Sigma, Castle Hill, New South 1; GFAP: glial fibrillary acidic protein; ANOVA: analysis of variance. Wales, Australia) or mouse anti-peripherin (1:400; Che- micon International, Billerica, MA, USA). Spinal cord Acknowledgements This work was supported by grants to GMT from the Faculty of Medicine, sections were stained for microglia with rabbit anti- The University of New South Wales, the National Health and Medical IBA1 (1:2000; Wako, Osaka, Japan) and for astrocytes Research Council of Australia (ID # 568637), and the NSW Office for Science with mouse anti-GFAP (1:2000; Chemicon Interna- and Medical Research. tional). Sections were blocked and then incubated with Author details the primary antibody. Elimination of the primary anti- School of Medical Sciences, University of New South Wales, Sydney, NSW body wasusedasanegative control. Thesections were 2052 Australia. Department of Neurobiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China. washed 4 times and incubated with a secondary anti- Department of Physiology, University of Sydney, NSW 2006 Australia. body as appropriate: donkey anti-mouse IgG conjugated with Cy2 (1:100; Jackson ImmunoResearch, West Grove, Authors’ contributions GM-T designed and coordinated the study, carried out some experiments, PA, USA) or donkey anti-rabbit IgG conjugated with analyzed data and wrote the manuscript. ML and HNA carried out some Cy3 (1:400; Jackson ImmunoResearch). In the case of behavioral testing, tissue processing and immunohistochemical analysis. 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Molecular Pain – Springer Journals

Published: Jan 5, 2011

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