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Spinal pathways involved in supraspinal modulation of neuropathic manifestations in rats
Pain 126 (2006) 280–293 www.elsevier.com/locate/pain
Spinal pathways involved in supraspinal modulation of neuropathic manifestations in rats Nayef E. Saade´ a
a,b,*
, Hassen Al Amin c, Steven Chalouhi a, Samah Abdel Baki a, Suhayl J. Jabbur b, Samir F. Atweh d
Department of Human Morphology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon b Department of Physiology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon c Department of Psychiatry, Faculty of Medicine, American University of Beirut, Beirut, Lebanon d Department of Internal Medicine, Faculty of Medicine, American University of Beirut, Beirut, Lebanon Received 5 February 2006; received in revised form 21 June 2006; accepted 17 July 2006
Abstract Controversial results have been recently reported on the role of supraspinal centers in the modulation of nociceptive behavior in animal models of mononeuropathy. Our aim was to investigate the role of the various spinal pathways in the modulation of the neuropathic manifestations. Several groups of rats were subjected to selective spinal-tract lesions, either 2–3 weeks before or 2–3 weeks after the induction of mononeuropathy following the chronic constriction injury (CCI) or the spared nerve injury (SNI) models. Tactile and cold allodynias were assessed by Von Frey filaments and the acetone drops test, respectively. Thermal hyperalgesia was assessed by the paw withdrawal and the hot plate tests. The effects of unilateral and bilateral lesions of the dorso-lateral funiculus (DLF), the anterolateral column (ALC) or hemisection were tested over a period of 4–8 weeks. All spinal tract lesions produced reversible, but significant decrease of allodynia and hyperalgesia over a period of 1–3 weeks. The most pronounced effects were observed with bilateral lesions. The stronger attenuation was observed on thermal hyperalgesia, assessed by the paw withdrawal test, while cold allodynia was the least affected. Spinal lesions performed before the induction of neuropathy did not produce significant alterations in the temporal development of neuropathic manifestations. The present results allow the conclusion that all spinal tracts can be involved in the rostral transmission and the descending modulation of neuropathic manifestations. The recovery of symptoms following spinal lesions provides illustration on the plasticity of the neural network involved in the processing of the neuropathic syndromes. 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Allodynia; Hyperalgesia; Peripheral neuropathy; Plasticity; Chronic pain
1. Introduction Injuries to the peripheral nerves can produce either transient or permanent structural and functional changes which have been reproduced in various experimental animal models (Bennett, 1994; Kim et al., 1997; Ralston, 1998). Among the important signs simulating neuro*
Corresponding author. Tel.: +961 1 350 000 4750; fax: +961 1 744464. E-mail address: [email protected] (N.E. Saade´).
pathic pain, the increased reactivity to nociceptive thermal stimuli has been considered as equivalent to thermal hyperalgesia, while the development of pronounced reactions to mild touch or to moderate cold has been considered as equivalent to mechanical and cold allodynias, respectively (Dowdall et al., 2005). Accumulating evidence attributes the triggering and the persistence of these signs to a cascade of events initiated by the injury to peripheral nerves. These include (1) functional (Wall and Gutnick, 1974; Campbell et al., 1988; Baron and Saguer, 1993; Devor and Seltzer,
0304-3959/$32.00 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2006.07.010
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1999) and phenotypic (Sato and Perl, 1991; Dib-Hajj et al., 1999; Ma et al., 2005) changes in the injured and the non-injured nerve fibers (Ali et al., 1999; Wu et al., 2001; Ma et al., 2003; Obata et al., 2003; Ringkamp and Meyer, 2005); (2) functional and structural changes in the dorsal horn neurons (McMahon and Wall, 1984; Gracely et al., 1992; Coderre et al., 1993; Goff et al., 1998; Shimoyama et al., 2005) and primary afferents (McMahon and Kett-White, 1991; Woolf et al., 1995; Goff et al., 1998; Bao et al., 2002); (3) alteration in the functions of the brainstem sensory (Persson et al., 1993; Miki et al., 1998; Day et al., 2001) and pain modulating centers (Pertovaara et al., 1997; Ossipov et al., 2001); (4) changes in the behavior of thalamic (Guilbaud et al., 1990; Iadarola et al., 1995; Miki et al., 2000; Bruggemann et al., 2001; Goettl et al., 2002) or cerebral cortical neurons (Mao et al., 1993; Backonja et al., 1994; Dougherty and Lenz, 1994). Despite almost unanimous agreement on the origin and functional significance of these manifestations, issues related to their supraspinal processing and to their persistence remain to be ascertained. As illustration, tactile allodynia has been claimed to depend on the dorsal column (DC)-medial lemniscus system (Miki et al., 1998; Sun et al., 2001) which also implies that the remaining neuropathic manifestations depend on other ascending fiber systems. However, the persistence of tactile allodynia and hyperalgesia appears to depend on the descending facilitation from the rostral ventromedial medulla (RVM) through the dorso-lateral funiculus (DLF) (Sung et al., 1998; Ossipov et al., 2000). Furthermore, it has also been suggested that mechanisms that initiate neuropathic manifestations are different from those involved in their persistence, which depend mainly on facilitatory influence descending from the brainstem (Burgess et al., 2002; Heinricher et al., 2003). Using two animal models for mononeuropathy, this study aimed at providing possible answers to the following questions: (1) what are the spinal tracts that are responsible for the processing of neuropathic manifestations? (2) Are there separate central tracts for the transmission of allodynia and hyperalgesia, like those observed in the peripheral nervous system? (3) What is the role of supraspinal centers in the initiation and maintenance of neuropathic manifestations? 2. Materials and methods All experiments were performed on adult Sprague–Dawley rats with an average weight of 250 g, housed in plastic transparent cages under standard colony conditions (12-h light–dark cycle, 22 ± 2 C and free access to food and water). Surgical procedures were performed under deep anesthesia made of atropine (0.05 mg/kg, i.p.) and chlorpromazine (8 mg/kg, i.p.) as preanesthesia which was followed by ketamine
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injection (Ketalar 50 mg/kg, i.p). The experimental procedures were approved by the Institutional Animal Care Committee and adhered to the ethical guidelines for pain experimentation on awake animals (Zimmermann, 1983). 2.1. Induction of mononeuropathy This was performed using either the chronic constriction injury (CCI) model (Bennett and Xie, 1988) or the spared nerve injury (SNI) model (Decosterd and Woolf, 2000), on the left hind paw of each rat. For the CCI model, four loose ligations (1 mm apart), using 4.0 chromic gut sutures, were made along the exposed sciatic nerve proximal to its trifurcation. For the SNI model, the tibial and common peroneal components of the sciatic nerve were carefully isolated, tightly ligated and sectioned while the sural nerve was left in place. At the end of surgical procedures, the wound was sutured in layers and the rats received prophylactic injection of antibiotic (Penicillin G, one million UI, s.c.) and were allowed to recover for a period of 3–5 days. 2.2. Spinal tract lesions The rat’s head was carefully placed in a stereotaxic apparatus and different spinal tract lesions were performed at either the C2–C3 level (upper cervical region) or the C7–C8 level (lower cervical region). After exposing the space between the cervical vertebrae with a minimal dorsal laminectomy and with the help of an operating microscope, the dura matter was sectioned and reflected, and jeweler’s forceps and fine scissors were used to lesion the spinal pathways. The blades of the forceps were pinched together to encompass the entire tract of interest and were held closed for a few seconds. Detailed description of spinal tract lesions was given previously (Wall et al., 1988; Saade´ et al., 1990). Hemisection and all bilateral tract lesions were performed at lower cervical level (C7–C8) to avoid any possible postoperative complications due to the extension of the damage or oedema to spinal centers or tracts involved in cardiac and respiratory regulations. For the anterolateral column (ALC) lesion, the tips of the forceps were introduced between the lateral edge of the spinal cord and the floor of the vertebral canal. The lesion required a mild rotation of the spinal cord exposing the exit of the ventral roots. Unilateral ALC lesion was performed at C2–C3 level, while bilateral lesions were made at C7–C8 level. Lesion of the dorsolateral funiculus (DLF) was made by introducing the tips of the forceps between the entry of the dorsal root and the lateral edge of the spinal cord, delineated at the C2–C3 level by the exit of the accessory nerve. For hemisection, the contralateral side of the spinal cord (with reference to the neuropathic paw) was tightly compressed between the tips of the forceps to produce clear interruption of the white columns at C7–C8 level. At the end of surgery, the exposed surface of the spinal cord was covered with a piece of Gelfoam and the wound was sutured in layers. The rats were given one injection of dexamethasone (2 mg/kg, i.p.) and daily injections of penicillin, during the first 2–3 days following the surgery.
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2.3. Experimental protocols This report is based on the results of observations made on two experimental sets. In the first set, spinal lesions were performed at least 2 weeks following the induction of neuropathy using the SNI model. This model is known to present a stable plateau of neuropathic manifestations over a period of few months. This stability allows to preclude any possible interference between the recovery from the effects of spinal cord lesions and the temporal alterations in the neuropathic manifestations (see details in Saade´ et al., 2002). This set includes one group (n = 5–6 rats) per each unilateral or bilateral lesion of the DLF, ALC and hemisection. Unilateral lesions were usually made at a high cervical level on the side opposite to the neuropathic paw. An additional group of rats was subjected to a combined ipsilateral lesion of the DLF and ALC. In a second experimental set, spinal lesions were performed first to be followed by a mononeuropathy after a minimum period of 2 weeks. Two models, the CCI and SNI, were used, since the main aim of this set was to assess whether spinal lesions can prevent, delay or attenuate the expression of neuropathic manifestations. This set includes one group per model and for each of the following lesions: bilateral DLF, contralateral ALC or DLF + ALC. A control group (n = 6) for the spinal lesions following the induction of neuropathy consisted in performing the cervical surgery in neuropathic rats and opening the dura mater but without spinal lesion. For the second experimental set, based on spinal lesions preceding neuropathy, each rat served as its own control since nociceptive thresholds were monitored for a minimum of 2 weeks before performing nerve lesion. 2.4. Behavioral tests Rats were brought to the experimental room, 3 days before any experimental intervention, to perform the baseline observations. All tests were carried out during the morning. Rats were placed individually in plastic boxes (16 · 18 · 17 cm) divided in two equal compartments, with a wire mesh floor supported by an elevated platform allowing clear observation and easy access to the plantar surface of the paw. The midplantar aspect of the hind paw was targeted in the CCI model, while the lateral plantar surface was targeted in the SNI model (i.e., the skin area innervated by the sural nerve). Before each test session, the animals were left for a period of 15–30 min allowing for adaptation to the environment. Tests were performed alternately and in a random order on the right (intact) and left (neuropathic) hind paws following the protocol described previously (Saade´ et al., 2002). Mechanical allodynia was assessed using Von Frey filaments (VF) of two different calibers corresponding to forces of 18.5 mN (2.04 g) and 106.7 mN (11.75 g). Each hair was applied perpendicularly upwards on the plantar surface of the paw with a force enough to bend the filament. Hair stimulations to the paw were applied first using the thin followed by the thick filament and ten trials, spaced by 10–15 s, were performed with each hair on each paw and the number of reactions was counted. Paw withdrawals due to locomotion or weight shifting were not counted. Both filaments are in the
innocuous range in control rats and are considered in the noxious range (allodynia) when eliciting P5 responses/10 trials in the neuropathic paw. Cold allodynia was measured according to the acetone drop test (Choi et al., 1994). Using a 1 ml syringe with a blunt tip needle, 50 ll of acetone was sprayed onto the plantar surface of the paw and the duration of paw withdrawal was measured, with 0.5 and 20 s assigned as minimum and maximum cut-off time points, respectively. Thermal hyperalgesia was assessed by using the paw withdrawal (PW) and the hot plate (HP) tests. For the PW test, a nociceptive radiant heat spot was applied on the plantar surface of the hind paws (Hargreaves et al., 1988) and the withdrawal latency (PWL) and duration (PWD) were recorded. Two trials, spaced by a minimum of 3 min, were performed on each paw and the recorded values for PWL and PWD were averaged. A maximum cut-off time was set at 14 s for the PWL test, whereas 0.5 and 10 s were taken as minimal and maximal cut-off points, respectively, for the PWD. For the HP test, rats were placed on a heated metal plate (53 ± 0.3 C) and the time the animal spent before licking the paw or leaping was recorded. Recovery from spinal lesions was assessed by the rotarod test, to measure the ability of animals to remain on a rotating cylinder with a constant rotation speed of approximately 5 revolutions/min. After an average of 1 week, all rats performed well on this test except for rats subjected to spinal hemisection or bilateral ALC lesion. The different tests were performed randomly in each session with a minimum of 5 min interval between two consecutive tests. All measurements were made by two independent observers, keeping two independent records that were averaged at the end of each session. One observer, at least, was blinded about the aims of the experiment and the performed experimental procedures. 2.5. Histology At the end of the observation period, rats were perfused intracardially, under deep anesthesia (pentobarbital 80– 100 mg/kg, i.p.), with phosphate-buffered saline (0.02 M PBS) containing heparin then with 10% formalin in 0.1 M PBS. The spinal cord was carefully isolated, removed and stored in jars containing 10% formalin for preservation and histological processing. After a minimum of 24 h, the spinal cord was sliced in 45 lm serial frozen sections and then stained with cresyl-violet. Microscopic inspection of the lesions was performed under light microscopy by a blinded observer who was not informed about the nature and the aims of the experimental procedures. Reported results in this study are based on observations made on rats with confirmed spinal lesions. 2.6. Data analysis After the induction of neuropathy, data obtained from behavioral tests, in each experimental group, on the intact and neuropathic paws, before and after the lesions, were averaged (mean ± SEM) for each time interval. The observed results for each time interval were compared to the controls established before the experimental procedures.
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The significance of the differences between experimental points and controls was calculated using ANOVA followed by Bonferroni post hoc test. Statistical analysis and graphs were made using GraphPad Instat and Prism packages (GraphPad, software, CA, USA).
3. Results 3.1. General observations All rats with spinal lesions showed rapid recovery within the first week after surgery. Their motor behavior was assessed twice a week by observing the normal exploration and grooming behavior and by subjecting each rat to the rotarod test (wooden cylinder 11 cm diameter rotating at a speed of 5 rpm). By the end of the first week after surgery, rats displayed normal spontaneous motor behavior and performed well on the rotarod test except for those subjected to hemisection or bilateral ALC lesions. Rats in these two last groups showed difficulties in alternating movements and could not perform for more than 10 s on this test. Part of this defect could be attributed to impairment of the movements of the forelimbs due to segmental effects of the lesion. However, reflex reactivity to nociceptive pinching of the lower limbs was normal or even exaggerated. Control rats, with sham cervical surgery, did not show significant alteration in neuropathic manifesta-
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tions, as compared to values recorded before surgery (data not shown). On the other hand, rats subjected to spinal lesion before mononeuropathy were allowed a minimum of 2 weeks before the induction of neuropathy. Their nociceptive thresholds were comparable in both hind paws (ipsi- and contralateral to spinal lesion) and also to values observed in control rats or in the same rats before the surgery. The dissipation of the effects of spinal lesions within 1 week after surgery and the recovery of normal nociceptive thresholds can be explained by the fact that the tests for nociception were performed on the hind paws supplied by spinal segments distant from the site of lesions. 3.2. Spinal tract lesion following the induction of mononeuropathy Two groups (n = 6 each) of rats were subjected either to a lesion of the DLF (C2–C3 level) contralateral to the neuropathic paw or to bilateral DLF lesions at C7–C8 level. Lesions were performed at the peak of neuropathic manifestations, i.e., 2 weeks following the nerve injury using the SNI model. Contralateral DLF lesion produced a short lived and moderate attenuation of tactile hyperreactivity and thermal hyperalgesia assessed by the PWD to heat (Fig. 1). All effects disappeared within a week after the lesion.
Fig. 1. Time courses of the effects of DLF lesions on neuropathic manifestations in rats subjected to SNI. The control in each panel represents the average of measurements performed on rats at the peak of neuropathic manifestations before the spinal lesion. One group (n = 6) was subjected to one side DLF lesion (opposite to neuropathic paw), while the second group received bilateral DLF lesions (n = 6). The significance of differences was measured with reference to the control using ANOVA followed by Bonferroni post hoc test. DLF, dorsolateral funiculus; VF, Von Frey filament; PWD, paw withdrawal duration; PWL, paw withdrawal latency. The abbreviations are used by the subsequent figures.
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Bilateral DLF lesions produced a significant and sustained decrease of neuropathic manifestations. Reactions of the neuropathic paw to the Von Frey filaments and to nociceptive heating spot became comparable to those of the intact paw. Furthermore, the hypersensitivity to the cold test (PWD/cold) displayed significant attenuation that was still more pronounced than that of the intact paw. This attenuation, however, decreased progressively and disappeared within 2–3 weeks following the lesion. The paw withdrawal latency and the hot plate test did not show significant alteration in their latencies (Fig. 1). The photomicrographs in Fig. 2 illustrate the extent of each DLF lesion. In both groups the damaged area of the white matter appears to correspond to the DLF tract. Another two groups (n = 6 each) of rats were subjected to either contralateral ALC lesion (with reference to the injured paw) at C2–C3 level or to bilateral ALC lesions at C7–C8 level. Unilateral ALC lesion produced
significant reduction of tactile and cold allodynias, that disappeared within 2 weeks (Fig. 3). Thermal hyperalgesia, however, showed more pronounced attenuation that lasted over a period of 3 weeks (Fig. 3, PWD/Heat and hot plate). Bilateral ALC lesions produced more pronounced attenuation of the hyperreactivity to tactile and cold stimuli which persisted for 2 and 3 weeks, respectively (Fig. 3, bilateral ALC). Heat hyperalgesia, assessed by the PWD and the HP tests, displayed the same evolution observed with unilateral ALC lesion (Fig. 3). Examination of histology sections revealed that unilateral ALC lesion involved, in most of the cases, all the fibers of the lateral columns and parts of the DLF (Fig. 4, Sections 2–4). The ventral column was spared in most of the cases except in rats 3 and 4 (Fig. 4). Bilateral lesions involved the lateral columns and the ventral aspect of the ventral quadrant. The photomicrographs shown in Fig. 4 illustrate the extent of the lesion on
Fig. 2. Photomicrographs of Nissl stained spinal sections at C2–C3 level in the contralateral DLF and at C7–C8 for bilateral DLF.
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Fig. 3. Time courses of the effects of anterolateral columns (ALC) lesion performed in two groups of rats (n = 6 each) at the peak of neuropathic manifestations using the SNI model.
one side but cannot account for the extent of lesions on both sides since it was difficult to have a section passing through the ideal plane of both lesions. However, the examination of serial section revealed the completeness of lesions on both sides of the spinal cord. Furthermore, all rats of the group displayed comparable evolutions of their motor and nociceptive behaviors. Hemisection of the spinal cord at C7–C8 was performed in one group of rats (n = 6) on the opposite side of the neuropathic paw. This lesion produced a significant (P < 0.001) reduction of tactile and cold allodynias over a period of 2 weeks; thermal hyperalgesia, assessed by the PWD test, was reduced for a more prolonged period (.4 weeks) while the hot plate test showed a shorter period of increased latency (or decreased hyperalgesia) (Fig. 5A). Microscopic examination of spinal cord sections revealed an almost total ablation of one side of the cord (Fig. 5B). Combined lesion to the DLF and ALC at C7–C8 levels ipsilateral to the neuropathic paw did not produce significant alteration in the neuropathic manifestations (Fig. 6A). Fig. 6B illustrates the completeness of the lesion which involved almost one side of the spinal cord minus the dorsal column. 3.3. Spinal tract lesions preceding the induction of mononeuropathy Six groups (n = 5–6 each) of rats were subjected to selective spinal tract lesions as 2 groups per each of the following: bilateral DLF, unilateral ALC and unilat-
eral DLF + ALC. After a recovery period of 2–3 weeks, each lesion was followed by the induction of mononeuropathy using either the CCI model (one group) or the SNI model (another group). As illustrated in Figs. 7–9, neuropathic manifestations displayed a normal temporal evolution with a normal time to onset and a maximum at 1–2 weeks following the nerve injury. Neuropathic manifestations maintained a constant plateau in rats subjected to SNI, while one group subjected to CCI neuropathy showed recovery after 6 weeks (Fig. 9). 4. Discussion The results of the present study can be summarized into four main observations. First, damage to any fiber tract in the spinal cord can lead to significant attenuation of the neuropathic-like behavior which was more pronounced and sustained following bilateral lesions. Second, the thermal hyperalgesia, assessed by the PWD test, was the most affected observation in all types of lesions, while the cold allodynia was the least affected to be followed by the tactile allodynia. Third, the effects of tract lesions were reversible within a period of 2–3 weeks. Fourth, spinal lesions performed prior to the induction of neuropathy did not prevent the development of neuropathic-like behavior. Lesions of the contralateral DLF and ALC are supposed to interrupt most of the somato-sensory inputs (which is mainly nociceptive) from neuropathic paw to the contralateral brainstem nuclei, thalamus,
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Fig. 4. Photomicrograph of Nissl stained spinal sections to illustrate the extent of ALC lesions in two different groups of rats.
hypothalamus and cerebral hemispheres. On the other hand, lesion of the ipsilateral spinal tracts may interrupt most of the descending control coming from the contralateral brain centers. However, accumulated evidence tends to demonstrate the existence of bilateral ascending and descending projections, although the contralateral component is more important in general. As illustration, lesions to the spinal tracts ipsilateral to the neuropathic paw did not produce significant effects (see Fig. 6, DLF + ALC), but their association with the section of the same tract in the contralateral side produced more significant effects than interrupting the contralateral tracts alone. This observation indicates the possible contribution of the ipsilateral spinal tracts in the neuropathic manifestations following alteration of the function of spinal tracts contralateral to the neuropathic paw. Therefore, the observed changes in neuropathic manifestations can be attributed to the interruption of supraspinal influences and cannot be due to spinal segmental changes since the
level of spinal lesion is located far from the segments supplying the neuropathic limb. The reported illustrations of spinal lesions represent, at best, the residual changes observed 4–8 weeks following the lesions, since it is well known that surgical procedures may result in a cascade of events involving breaking of blood–brain barrier, immune cell infiltration, œdema, ischemic changes and inflammatory reaction. Such events can affect an area in the spinal cord greater than that observed on histological section and may contribute, despite treatment with anti-inflammatory drugs and antibiotics, to the attenuation of neuropathic manifestations. Therefore, one might assume that the results of spinal lesions, performed after the induction of neuropathy, are due to the association of the effects of interruption of a specific tract to the trauma that can affect the general function of the spinal cord. However, the persistence of the effects over a period longer than that needed for recovery may give indication on the role of the lesioned tract as it was previously
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Fig. 5. Time courses of the effects of contralateral hemisection on neuropathic manifestations in rats subjected to SNI mononeuropathy (A). (B) Selected photographs of Nissl stained spinal sections to illustrate the extent of the lesion in each rat.
described (Saade´ et al., 2002). Furthermore, the mild effects observed following lesions of the ipsilateral DLF, ALC or their combination may account for the effects of the trauma and provide evidence on the specific effects of these tracts when the lesion is made on the opposite side of the neuropathic paw. Further information about the role of injury and recovery is gathered from the study of the effects of spinal tracts, lesions prior to the induction of mononeuropathy. The lack of effects of lesions to DLF, ALC or both, when performed 2 weeks prior to nerve lesion, can be simply attributed to the recovery from the damage due to the trauma and the interruption of spinal tracts.
These results correlate well with our previous observations on the effects of dorsal column lesions on neuropathic manifestations when performed at various timings before, simultaneous, with or after the induction of mononeuropathy (Saade´ et al., 2002). Thus, it appears that injury to a spinal tract does not prevent the development of neuropathic manifestations, which implies that signalling of these manifestations can be made or rerouted through the available intact spinal tracts. Several studies, using either the CCI or the spinal nerve ligation (SNL) model, have described supraspinal modulation of the neuropathic-like behavior in rats. As
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Fig. 6. Time courses of the effects of combined DLF + ALC lesions ipsilateral to the neuropathic paw (A). Nissl stained spinal sections (B) illustrate the extent of the lesions in each rat.
illustration, spinal lesions have been shown to abolish tactile allodynia without altering heat hyperalgesia (Bian et al., 1998; Sung et al., 1998). Subsequent investigations have shown that tactile allodynia depends exclusively on the transmission through the dorsal columns (Miki et al., 2000; Sun et al., 2001). However, previous studies by our group, using the SNI and CCI models, have provided evidences for the following conclusions: (1) Dorsal column lesions can reduce all the neuropathic manifestations including tactile and cold allodynia and heat hyperalgesia. (2) Long-term observations have revealed recurrence of all neuropathic manifestations previously depressed by DC lesion (Saade´ et al., 2002).
Although the large fiber afferents are known to be involved in tactile allodynia (Cervero and Laird, 1996; Miki et al., 1998; Ossipov et al., 1999; Sun et al., 2001; Pitcher and Henry, 2004), several recent investigations have reported that the maintenance of allodynia and hyperalgesia depends on descending facilitatory influences from the brainstem through the ipsilateral DLF (Kovelowski et al., 2000; Ossipov et al., 2000; Azami et al., 2001; Porreca et al., 2002). Thus, the neuropathic manifestations appear to depend on a spinal–brainstem– spinal loop with an ascending component in the DC and a descending component in the DLF ipsilateral to the injured paw (Burgess et al., 2002; Porreca et al., 2002). The results of the present study demonstrate that lesions
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Fig. 7. Time courses of the development of neuropathic manifestations in rats sustaining chronic bilateral DLF lesions at C7–C8 levels. Nerve injury was performed using the CCI and the SNI models (one group per model) in two separate groups of rats (n = 6) after complete recovery from the surgery for DLF lesion.
Fig. 8. Time courses of the development of neuropathic manifestations in rats sustaining chronic contralateral ALC lesions at C7–C8 levels. Nerve injury was performed using the CCI and the SNI models (one group per model) in two separate groups of rats (n = 6) after complete recovery from the surgery for ALC lesion.
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Fig. 9. Time courses of the development of neuropathic manifestations in rats subjected to chronic combined DLF + ALC lesions placed at C7–C8 levels contralateral to the neuropathic paw. Nerve injury was performed using the CCI and the SNI models (one group per model) in two separate groups of rats (n = 6) after complete recovery from the surgery for DLF + ALC lesions.
to either the ipsilateral or contralateral DLF did not abolish the neuropathic manifestations and that allodynia and hyperalgesia developed normally in rats sustaining chronic DLF or ALC lesions prior to the induction of neuropathy. Therefore, our results do not support the hypothesis of descending facilitation. In addition, the observed recurrence of neuropathic manifestations appears to be more in line with the original concept of descending inhibitory influences on nociception from brainstem centers (Basbaum and Fields, 1978, 1984). Moreover, other studies reported that neuropathy can activate inhibitory mechanisms of nociception descending in the DLF system (Wall et al., 1988; Sotgiu, 1993; Danziger et al., 2001; Ma and Eisenach, 2003). Apart from some differential effects, our results have revealed that all spinal tract lesions can attenuate tactile allodynia and thermal hyperalgesia. These findings provide clear evidence against the concept of modality-specific segregation of the spinal pathways involved in rostral modulation or signalling of neuropathic manifestations (Bester et al., 2000; Blomqvist and Craig, 2000), since lesion of each tract was able to produce reversible attenuation of all neuropathic manifestations and that these manifestations showed regular development in rats sustaining various spinal tract lesions prior to the induction of mononeuropathies. The observed recurrence of the neuropathic manifestations complements the idea
of plasticity of channeling of nociception through the available pathway. This hypothesis correlates well with clinical observations reporting that under pathological conditions, nociceptive signalling can travel through the DC system (Triggs and Beric, 1994; Beric, 1997). It can be also correlated with clinical observations reporting the failure of cordotomies in relieving chronic pains (Nathan and Smith, 1979; Gybels and Tasker, 1999). Our results showed consistently reversible attenuation of the neuropathic manifestations which reflects a transient interruption of either facilitatory or inhibitory supraspinal control mechanisms. This assumption is supported by previous reports from our laboratory showing short- and long-term modulation of neuropathic-like behavior by interfering with the normal function of the ventral orbital area (Baliki et al., 2003; Al-Amin et al., 2004) or the somatosensory thalamic nuclei (Saade´ et al., 2006). However, there remains the need to explain the following two observations: Why were all the observed effects involving only attenuation of the neuropathic manifestations? Why was attenuation reversible and followed by a full recurrence of these manifestations? Unlike the autotomy model (Wall et al., 1988; Saade´ et al., 1990) spinal lesions (DLF mainly) did not increase the neuropathic manifestations, perhaps, because these manifestations are already set at their maximal level and the lack of attenuation can be
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considered as equivalent to facilitation. The recovery of manifestations following each lesion may indicate a delayed adjustment to the loss of supraspinal influences. A final question that emanates from this discussion: are these manifestations simple spinal reflexes or elements of a more elaborate behavior related to chronic pain? A possible answer is that each of these manifestations depends on a spinal circuitry, but this circuitry is part of spinal and supraspinal network involved in the processing of nociceptive information and the organization of pain-related behavior. Finally, the results of the present study lead to the following conclusions. First, the observed tactile and cold allodynia and heat hyperalgesia are not simple spinal reflexes, but depend to a large extent on supraspinal influences. Second, the reversible attenuation of neuropathic manifestations, produced by selective lesions of the various spinal tracts, provides evidence about the importance of the supraspinal modulatory mechanisms and on the flexibility of the neural network involved in this process. Third, the observed involvement of all the spinal tracts in the modulation of the allodynia and thermal hyperalgesia provides evidence against the concept of specific fiber tracts involved in the processing of the different aspects of neuropathic manifestations.
Acknowledgements The authors thank Sawsan Sharrouf, Bilal Mazeh, Bassem Najm, Nadine Fawaz, and Riad Maalouf for their technical assistance. This project was supported by grant from the Lebanese National Council for Scientific Research.
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Spinal pathways involved in supraspinal modulation of neuropathic manifestations in rats Nayef E. Saade´ a
a,b,*
, Hassen Al Amin c, Steven Chalouhi a, Samah Abdel Baki a, Suhayl J. Jabbur b, Samir F. Atweh d
Department of Human Morphology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon b Department of Physiology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon c Department of Psychiatry, Faculty of Medicine, American University of Beirut, Beirut, Lebanon d Department of Internal Medicine, Faculty of Medicine, American University of Beirut, Beirut, Lebanon Received 5 February 2006; received in revised form 21 June 2006; accepted 17 July 2006
Abstract Controversial results have been recently reported on the role of supraspinal centers in the modulation of nociceptive behavior in animal models of mononeuropathy. Our aim was to investigate the role of the various spinal pathways in the modulation of the neuropathic manifestations. Several groups of rats were subjected to selective spinal-tract lesions, either 2–3 weeks before or 2–3 weeks after the induction of mononeuropathy following the chronic constriction injury (CCI) or the spared nerve injury (SNI) models. Tactile and cold allodynias were assessed by Von Frey filaments and the acetone drops test, respectively. Thermal hyperalgesia was assessed by the paw withdrawal and the hot plate tests. The effects of unilateral and bilateral lesions of the dorso-lateral funiculus (DLF), the anterolateral column (ALC) or hemisection were tested over a period of 4–8 weeks. All spinal tract lesions produced reversible, but significant decrease of allodynia and hyperalgesia over a period of 1–3 weeks. The most pronounced effects were observed with bilateral lesions. The stronger attenuation was observed on thermal hyperalgesia, assessed by the paw withdrawal test, while cold allodynia was the least affected. Spinal lesions performed before the induction of neuropathy did not produce significant alterations in the temporal development of neuropathic manifestations. The present results allow the conclusion that all spinal tracts can be involved in the rostral transmission and the descending modulation of neuropathic manifestations. The recovery of symptoms following spinal lesions provides illustration on the plasticity of the neural network involved in the processing of the neuropathic syndromes. 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Allodynia; Hyperalgesia; Peripheral neuropathy; Plasticity; Chronic pain
1. Introduction Injuries to the peripheral nerves can produce either transient or permanent structural and functional changes which have been reproduced in various experimental animal models (Bennett, 1994; Kim et al., 1997; Ralston, 1998). Among the important signs simulating neuro*
Corresponding author. Tel.: +961 1 350 000 4750; fax: +961 1 744464. E-mail address: [email protected] (N.E. Saade´).
pathic pain, the increased reactivity to nociceptive thermal stimuli has been considered as equivalent to thermal hyperalgesia, while the development of pronounced reactions to mild touch or to moderate cold has been considered as equivalent to mechanical and cold allodynias, respectively (Dowdall et al., 2005). Accumulating evidence attributes the triggering and the persistence of these signs to a cascade of events initiated by the injury to peripheral nerves. These include (1) functional (Wall and Gutnick, 1974; Campbell et al., 1988; Baron and Saguer, 1993; Devor and Seltzer,
0304-3959/$32.00 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2006.07.010
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1999) and phenotypic (Sato and Perl, 1991; Dib-Hajj et al., 1999; Ma et al., 2005) changes in the injured and the non-injured nerve fibers (Ali et al., 1999; Wu et al., 2001; Ma et al., 2003; Obata et al., 2003; Ringkamp and Meyer, 2005); (2) functional and structural changes in the dorsal horn neurons (McMahon and Wall, 1984; Gracely et al., 1992; Coderre et al., 1993; Goff et al., 1998; Shimoyama et al., 2005) and primary afferents (McMahon and Kett-White, 1991; Woolf et al., 1995; Goff et al., 1998; Bao et al., 2002); (3) alteration in the functions of the brainstem sensory (Persson et al., 1993; Miki et al., 1998; Day et al., 2001) and pain modulating centers (Pertovaara et al., 1997; Ossipov et al., 2001); (4) changes in the behavior of thalamic (Guilbaud et al., 1990; Iadarola et al., 1995; Miki et al., 2000; Bruggemann et al., 2001; Goettl et al., 2002) or cerebral cortical neurons (Mao et al., 1993; Backonja et al., 1994; Dougherty and Lenz, 1994). Despite almost unanimous agreement on the origin and functional significance of these manifestations, issues related to their supraspinal processing and to their persistence remain to be ascertained. As illustration, tactile allodynia has been claimed to depend on the dorsal column (DC)-medial lemniscus system (Miki et al., 1998; Sun et al., 2001) which also implies that the remaining neuropathic manifestations depend on other ascending fiber systems. However, the persistence of tactile allodynia and hyperalgesia appears to depend on the descending facilitation from the rostral ventromedial medulla (RVM) through the dorso-lateral funiculus (DLF) (Sung et al., 1998; Ossipov et al., 2000). Furthermore, it has also been suggested that mechanisms that initiate neuropathic manifestations are different from those involved in their persistence, which depend mainly on facilitatory influence descending from the brainstem (Burgess et al., 2002; Heinricher et al., 2003). Using two animal models for mononeuropathy, this study aimed at providing possible answers to the following questions: (1) what are the spinal tracts that are responsible for the processing of neuropathic manifestations? (2) Are there separate central tracts for the transmission of allodynia and hyperalgesia, like those observed in the peripheral nervous system? (3) What is the role of supraspinal centers in the initiation and maintenance of neuropathic manifestations? 2. Materials and methods All experiments were performed on adult Sprague–Dawley rats with an average weight of 250 g, housed in plastic transparent cages under standard colony conditions (12-h light–dark cycle, 22 ± 2 C and free access to food and water). Surgical procedures were performed under deep anesthesia made of atropine (0.05 mg/kg, i.p.) and chlorpromazine (8 mg/kg, i.p.) as preanesthesia which was followed by ketamine
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injection (Ketalar 50 mg/kg, i.p). The experimental procedures were approved by the Institutional Animal Care Committee and adhered to the ethical guidelines for pain experimentation on awake animals (Zimmermann, 1983). 2.1. Induction of mononeuropathy This was performed using either the chronic constriction injury (CCI) model (Bennett and Xie, 1988) or the spared nerve injury (SNI) model (Decosterd and Woolf, 2000), on the left hind paw of each rat. For the CCI model, four loose ligations (1 mm apart), using 4.0 chromic gut sutures, were made along the exposed sciatic nerve proximal to its trifurcation. For the SNI model, the tibial and common peroneal components of the sciatic nerve were carefully isolated, tightly ligated and sectioned while the sural nerve was left in place. At the end of surgical procedures, the wound was sutured in layers and the rats received prophylactic injection of antibiotic (Penicillin G, one million UI, s.c.) and were allowed to recover for a period of 3–5 days. 2.2. Spinal tract lesions The rat’s head was carefully placed in a stereotaxic apparatus and different spinal tract lesions were performed at either the C2–C3 level (upper cervical region) or the C7–C8 level (lower cervical region). After exposing the space between the cervical vertebrae with a minimal dorsal laminectomy and with the help of an operating microscope, the dura matter was sectioned and reflected, and jeweler’s forceps and fine scissors were used to lesion the spinal pathways. The blades of the forceps were pinched together to encompass the entire tract of interest and were held closed for a few seconds. Detailed description of spinal tract lesions was given previously (Wall et al., 1988; Saade´ et al., 1990). Hemisection and all bilateral tract lesions were performed at lower cervical level (C7–C8) to avoid any possible postoperative complications due to the extension of the damage or oedema to spinal centers or tracts involved in cardiac and respiratory regulations. For the anterolateral column (ALC) lesion, the tips of the forceps were introduced between the lateral edge of the spinal cord and the floor of the vertebral canal. The lesion required a mild rotation of the spinal cord exposing the exit of the ventral roots. Unilateral ALC lesion was performed at C2–C3 level, while bilateral lesions were made at C7–C8 level. Lesion of the dorsolateral funiculus (DLF) was made by introducing the tips of the forceps between the entry of the dorsal root and the lateral edge of the spinal cord, delineated at the C2–C3 level by the exit of the accessory nerve. For hemisection, the contralateral side of the spinal cord (with reference to the neuropathic paw) was tightly compressed between the tips of the forceps to produce clear interruption of the white columns at C7–C8 level. At the end of surgery, the exposed surface of the spinal cord was covered with a piece of Gelfoam and the wound was sutured in layers. The rats were given one injection of dexamethasone (2 mg/kg, i.p.) and daily injections of penicillin, during the first 2–3 days following the surgery.
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2.3. Experimental protocols This report is based on the results of observations made on two experimental sets. In the first set, spinal lesions were performed at least 2 weeks following the induction of neuropathy using the SNI model. This model is known to present a stable plateau of neuropathic manifestations over a period of few months. This stability allows to preclude any possible interference between the recovery from the effects of spinal cord lesions and the temporal alterations in the neuropathic manifestations (see details in Saade´ et al., 2002). This set includes one group (n = 5–6 rats) per each unilateral or bilateral lesion of the DLF, ALC and hemisection. Unilateral lesions were usually made at a high cervical level on the side opposite to the neuropathic paw. An additional group of rats was subjected to a combined ipsilateral lesion of the DLF and ALC. In a second experimental set, spinal lesions were performed first to be followed by a mononeuropathy after a minimum period of 2 weeks. Two models, the CCI and SNI, were used, since the main aim of this set was to assess whether spinal lesions can prevent, delay or attenuate the expression of neuropathic manifestations. This set includes one group per model and for each of the following lesions: bilateral DLF, contralateral ALC or DLF + ALC. A control group (n = 6) for the spinal lesions following the induction of neuropathy consisted in performing the cervical surgery in neuropathic rats and opening the dura mater but without spinal lesion. For the second experimental set, based on spinal lesions preceding neuropathy, each rat served as its own control since nociceptive thresholds were monitored for a minimum of 2 weeks before performing nerve lesion. 2.4. Behavioral tests Rats were brought to the experimental room, 3 days before any experimental intervention, to perform the baseline observations. All tests were carried out during the morning. Rats were placed individually in plastic boxes (16 · 18 · 17 cm) divided in two equal compartments, with a wire mesh floor supported by an elevated platform allowing clear observation and easy access to the plantar surface of the paw. The midplantar aspect of the hind paw was targeted in the CCI model, while the lateral plantar surface was targeted in the SNI model (i.e., the skin area innervated by the sural nerve). Before each test session, the animals were left for a period of 15–30 min allowing for adaptation to the environment. Tests were performed alternately and in a random order on the right (intact) and left (neuropathic) hind paws following the protocol described previously (Saade´ et al., 2002). Mechanical allodynia was assessed using Von Frey filaments (VF) of two different calibers corresponding to forces of 18.5 mN (2.04 g) and 106.7 mN (11.75 g). Each hair was applied perpendicularly upwards on the plantar surface of the paw with a force enough to bend the filament. Hair stimulations to the paw were applied first using the thin followed by the thick filament and ten trials, spaced by 10–15 s, were performed with each hair on each paw and the number of reactions was counted. Paw withdrawals due to locomotion or weight shifting were not counted. Both filaments are in the
innocuous range in control rats and are considered in the noxious range (allodynia) when eliciting P5 responses/10 trials in the neuropathic paw. Cold allodynia was measured according to the acetone drop test (Choi et al., 1994). Using a 1 ml syringe with a blunt tip needle, 50 ll of acetone was sprayed onto the plantar surface of the paw and the duration of paw withdrawal was measured, with 0.5 and 20 s assigned as minimum and maximum cut-off time points, respectively. Thermal hyperalgesia was assessed by using the paw withdrawal (PW) and the hot plate (HP) tests. For the PW test, a nociceptive radiant heat spot was applied on the plantar surface of the hind paws (Hargreaves et al., 1988) and the withdrawal latency (PWL) and duration (PWD) were recorded. Two trials, spaced by a minimum of 3 min, were performed on each paw and the recorded values for PWL and PWD were averaged. A maximum cut-off time was set at 14 s for the PWL test, whereas 0.5 and 10 s were taken as minimal and maximal cut-off points, respectively, for the PWD. For the HP test, rats were placed on a heated metal plate (53 ± 0.3 C) and the time the animal spent before licking the paw or leaping was recorded. Recovery from spinal lesions was assessed by the rotarod test, to measure the ability of animals to remain on a rotating cylinder with a constant rotation speed of approximately 5 revolutions/min. After an average of 1 week, all rats performed well on this test except for rats subjected to spinal hemisection or bilateral ALC lesion. The different tests were performed randomly in each session with a minimum of 5 min interval between two consecutive tests. All measurements were made by two independent observers, keeping two independent records that were averaged at the end of each session. One observer, at least, was blinded about the aims of the experiment and the performed experimental procedures. 2.5. Histology At the end of the observation period, rats were perfused intracardially, under deep anesthesia (pentobarbital 80– 100 mg/kg, i.p.), with phosphate-buffered saline (0.02 M PBS) containing heparin then with 10% formalin in 0.1 M PBS. The spinal cord was carefully isolated, removed and stored in jars containing 10% formalin for preservation and histological processing. After a minimum of 24 h, the spinal cord was sliced in 45 lm serial frozen sections and then stained with cresyl-violet. Microscopic inspection of the lesions was performed under light microscopy by a blinded observer who was not informed about the nature and the aims of the experimental procedures. Reported results in this study are based on observations made on rats with confirmed spinal lesions. 2.6. Data analysis After the induction of neuropathy, data obtained from behavioral tests, in each experimental group, on the intact and neuropathic paws, before and after the lesions, were averaged (mean ± SEM) for each time interval. The observed results for each time interval were compared to the controls established before the experimental procedures.
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The significance of the differences between experimental points and controls was calculated using ANOVA followed by Bonferroni post hoc test. Statistical analysis and graphs were made using GraphPad Instat and Prism packages (GraphPad, software, CA, USA).
3. Results 3.1. General observations All rats with spinal lesions showed rapid recovery within the first week after surgery. Their motor behavior was assessed twice a week by observing the normal exploration and grooming behavior and by subjecting each rat to the rotarod test (wooden cylinder 11 cm diameter rotating at a speed of 5 rpm). By the end of the first week after surgery, rats displayed normal spontaneous motor behavior and performed well on the rotarod test except for those subjected to hemisection or bilateral ALC lesions. Rats in these two last groups showed difficulties in alternating movements and could not perform for more than 10 s on this test. Part of this defect could be attributed to impairment of the movements of the forelimbs due to segmental effects of the lesion. However, reflex reactivity to nociceptive pinching of the lower limbs was normal or even exaggerated. Control rats, with sham cervical surgery, did not show significant alteration in neuropathic manifesta-
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tions, as compared to values recorded before surgery (data not shown). On the other hand, rats subjected to spinal lesion before mononeuropathy were allowed a minimum of 2 weeks before the induction of neuropathy. Their nociceptive thresholds were comparable in both hind paws (ipsi- and contralateral to spinal lesion) and also to values observed in control rats or in the same rats before the surgery. The dissipation of the effects of spinal lesions within 1 week after surgery and the recovery of normal nociceptive thresholds can be explained by the fact that the tests for nociception were performed on the hind paws supplied by spinal segments distant from the site of lesions. 3.2. Spinal tract lesion following the induction of mononeuropathy Two groups (n = 6 each) of rats were subjected either to a lesion of the DLF (C2–C3 level) contralateral to the neuropathic paw or to bilateral DLF lesions at C7–C8 level. Lesions were performed at the peak of neuropathic manifestations, i.e., 2 weeks following the nerve injury using the SNI model. Contralateral DLF lesion produced a short lived and moderate attenuation of tactile hyperreactivity and thermal hyperalgesia assessed by the PWD to heat (Fig. 1). All effects disappeared within a week after the lesion.
Fig. 1. Time courses of the effects of DLF lesions on neuropathic manifestations in rats subjected to SNI. The control in each panel represents the average of measurements performed on rats at the peak of neuropathic manifestations before the spinal lesion. One group (n = 6) was subjected to one side DLF lesion (opposite to neuropathic paw), while the second group received bilateral DLF lesions (n = 6). The significance of differences was measured with reference to the control using ANOVA followed by Bonferroni post hoc test. DLF, dorsolateral funiculus; VF, Von Frey filament; PWD, paw withdrawal duration; PWL, paw withdrawal latency. The abbreviations are used by the subsequent figures.
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Bilateral DLF lesions produced a significant and sustained decrease of neuropathic manifestations. Reactions of the neuropathic paw to the Von Frey filaments and to nociceptive heating spot became comparable to those of the intact paw. Furthermore, the hypersensitivity to the cold test (PWD/cold) displayed significant attenuation that was still more pronounced than that of the intact paw. This attenuation, however, decreased progressively and disappeared within 2–3 weeks following the lesion. The paw withdrawal latency and the hot plate test did not show significant alteration in their latencies (Fig. 1). The photomicrographs in Fig. 2 illustrate the extent of each DLF lesion. In both groups the damaged area of the white matter appears to correspond to the DLF tract. Another two groups (n = 6 each) of rats were subjected to either contralateral ALC lesion (with reference to the injured paw) at C2–C3 level or to bilateral ALC lesions at C7–C8 level. Unilateral ALC lesion produced
significant reduction of tactile and cold allodynias, that disappeared within 2 weeks (Fig. 3). Thermal hyperalgesia, however, showed more pronounced attenuation that lasted over a period of 3 weeks (Fig. 3, PWD/Heat and hot plate). Bilateral ALC lesions produced more pronounced attenuation of the hyperreactivity to tactile and cold stimuli which persisted for 2 and 3 weeks, respectively (Fig. 3, bilateral ALC). Heat hyperalgesia, assessed by the PWD and the HP tests, displayed the same evolution observed with unilateral ALC lesion (Fig. 3). Examination of histology sections revealed that unilateral ALC lesion involved, in most of the cases, all the fibers of the lateral columns and parts of the DLF (Fig. 4, Sections 2–4). The ventral column was spared in most of the cases except in rats 3 and 4 (Fig. 4). Bilateral lesions involved the lateral columns and the ventral aspect of the ventral quadrant. The photomicrographs shown in Fig. 4 illustrate the extent of the lesion on
Fig. 2. Photomicrographs of Nissl stained spinal sections at C2–C3 level in the contralateral DLF and at C7–C8 for bilateral DLF.
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Fig. 3. Time courses of the effects of anterolateral columns (ALC) lesion performed in two groups of rats (n = 6 each) at the peak of neuropathic manifestations using the SNI model.
one side but cannot account for the extent of lesions on both sides since it was difficult to have a section passing through the ideal plane of both lesions. However, the examination of serial section revealed the completeness of lesions on both sides of the spinal cord. Furthermore, all rats of the group displayed comparable evolutions of their motor and nociceptive behaviors. Hemisection of the spinal cord at C7–C8 was performed in one group of rats (n = 6) on the opposite side of the neuropathic paw. This lesion produced a significant (P < 0.001) reduction of tactile and cold allodynias over a period of 2 weeks; thermal hyperalgesia, assessed by the PWD test, was reduced for a more prolonged period (.4 weeks) while the hot plate test showed a shorter period of increased latency (or decreased hyperalgesia) (Fig. 5A). Microscopic examination of spinal cord sections revealed an almost total ablation of one side of the cord (Fig. 5B). Combined lesion to the DLF and ALC at C7–C8 levels ipsilateral to the neuropathic paw did not produce significant alteration in the neuropathic manifestations (Fig. 6A). Fig. 6B illustrates the completeness of the lesion which involved almost one side of the spinal cord minus the dorsal column. 3.3. Spinal tract lesions preceding the induction of mononeuropathy Six groups (n = 5–6 each) of rats were subjected to selective spinal tract lesions as 2 groups per each of the following: bilateral DLF, unilateral ALC and unilat-
eral DLF + ALC. After a recovery period of 2–3 weeks, each lesion was followed by the induction of mononeuropathy using either the CCI model (one group) or the SNI model (another group). As illustrated in Figs. 7–9, neuropathic manifestations displayed a normal temporal evolution with a normal time to onset and a maximum at 1–2 weeks following the nerve injury. Neuropathic manifestations maintained a constant plateau in rats subjected to SNI, while one group subjected to CCI neuropathy showed recovery after 6 weeks (Fig. 9). 4. Discussion The results of the present study can be summarized into four main observations. First, damage to any fiber tract in the spinal cord can lead to significant attenuation of the neuropathic-like behavior which was more pronounced and sustained following bilateral lesions. Second, the thermal hyperalgesia, assessed by the PWD test, was the most affected observation in all types of lesions, while the cold allodynia was the least affected to be followed by the tactile allodynia. Third, the effects of tract lesions were reversible within a period of 2–3 weeks. Fourth, spinal lesions performed prior to the induction of neuropathy did not prevent the development of neuropathic-like behavior. Lesions of the contralateral DLF and ALC are supposed to interrupt most of the somato-sensory inputs (which is mainly nociceptive) from neuropathic paw to the contralateral brainstem nuclei, thalamus,
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Fig. 4. Photomicrograph of Nissl stained spinal sections to illustrate the extent of ALC lesions in two different groups of rats.
hypothalamus and cerebral hemispheres. On the other hand, lesion of the ipsilateral spinal tracts may interrupt most of the descending control coming from the contralateral brain centers. However, accumulated evidence tends to demonstrate the existence of bilateral ascending and descending projections, although the contralateral component is more important in general. As illustration, lesions to the spinal tracts ipsilateral to the neuropathic paw did not produce significant effects (see Fig. 6, DLF + ALC), but their association with the section of the same tract in the contralateral side produced more significant effects than interrupting the contralateral tracts alone. This observation indicates the possible contribution of the ipsilateral spinal tracts in the neuropathic manifestations following alteration of the function of spinal tracts contralateral to the neuropathic paw. Therefore, the observed changes in neuropathic manifestations can be attributed to the interruption of supraspinal influences and cannot be due to spinal segmental changes since the
level of spinal lesion is located far from the segments supplying the neuropathic limb. The reported illustrations of spinal lesions represent, at best, the residual changes observed 4–8 weeks following the lesions, since it is well known that surgical procedures may result in a cascade of events involving breaking of blood–brain barrier, immune cell infiltration, œdema, ischemic changes and inflammatory reaction. Such events can affect an area in the spinal cord greater than that observed on histological section and may contribute, despite treatment with anti-inflammatory drugs and antibiotics, to the attenuation of neuropathic manifestations. Therefore, one might assume that the results of spinal lesions, performed after the induction of neuropathy, are due to the association of the effects of interruption of a specific tract to the trauma that can affect the general function of the spinal cord. However, the persistence of the effects over a period longer than that needed for recovery may give indication on the role of the lesioned tract as it was previously
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Fig. 5. Time courses of the effects of contralateral hemisection on neuropathic manifestations in rats subjected to SNI mononeuropathy (A). (B) Selected photographs of Nissl stained spinal sections to illustrate the extent of the lesion in each rat.
described (Saade´ et al., 2002). Furthermore, the mild effects observed following lesions of the ipsilateral DLF, ALC or their combination may account for the effects of the trauma and provide evidence on the specific effects of these tracts when the lesion is made on the opposite side of the neuropathic paw. Further information about the role of injury and recovery is gathered from the study of the effects of spinal tracts, lesions prior to the induction of mononeuropathy. The lack of effects of lesions to DLF, ALC or both, when performed 2 weeks prior to nerve lesion, can be simply attributed to the recovery from the damage due to the trauma and the interruption of spinal tracts.
These results correlate well with our previous observations on the effects of dorsal column lesions on neuropathic manifestations when performed at various timings before, simultaneous, with or after the induction of mononeuropathy (Saade´ et al., 2002). Thus, it appears that injury to a spinal tract does not prevent the development of neuropathic manifestations, which implies that signalling of these manifestations can be made or rerouted through the available intact spinal tracts. Several studies, using either the CCI or the spinal nerve ligation (SNL) model, have described supraspinal modulation of the neuropathic-like behavior in rats. As
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Fig. 6. Time courses of the effects of combined DLF + ALC lesions ipsilateral to the neuropathic paw (A). Nissl stained spinal sections (B) illustrate the extent of the lesions in each rat.
illustration, spinal lesions have been shown to abolish tactile allodynia without altering heat hyperalgesia (Bian et al., 1998; Sung et al., 1998). Subsequent investigations have shown that tactile allodynia depends exclusively on the transmission through the dorsal columns (Miki et al., 2000; Sun et al., 2001). However, previous studies by our group, using the SNI and CCI models, have provided evidences for the following conclusions: (1) Dorsal column lesions can reduce all the neuropathic manifestations including tactile and cold allodynia and heat hyperalgesia. (2) Long-term observations have revealed recurrence of all neuropathic manifestations previously depressed by DC lesion (Saade´ et al., 2002).
Although the large fiber afferents are known to be involved in tactile allodynia (Cervero and Laird, 1996; Miki et al., 1998; Ossipov et al., 1999; Sun et al., 2001; Pitcher and Henry, 2004), several recent investigations have reported that the maintenance of allodynia and hyperalgesia depends on descending facilitatory influences from the brainstem through the ipsilateral DLF (Kovelowski et al., 2000; Ossipov et al., 2000; Azami et al., 2001; Porreca et al., 2002). Thus, the neuropathic manifestations appear to depend on a spinal–brainstem– spinal loop with an ascending component in the DC and a descending component in the DLF ipsilateral to the injured paw (Burgess et al., 2002; Porreca et al., 2002). The results of the present study demonstrate that lesions
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Fig. 7. Time courses of the development of neuropathic manifestations in rats sustaining chronic bilateral DLF lesions at C7–C8 levels. Nerve injury was performed using the CCI and the SNI models (one group per model) in two separate groups of rats (n = 6) after complete recovery from the surgery for DLF lesion.
Fig. 8. Time courses of the development of neuropathic manifestations in rats sustaining chronic contralateral ALC lesions at C7–C8 levels. Nerve injury was performed using the CCI and the SNI models (one group per model) in two separate groups of rats (n = 6) after complete recovery from the surgery for ALC lesion.
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Fig. 9. Time courses of the development of neuropathic manifestations in rats subjected to chronic combined DLF + ALC lesions placed at C7–C8 levels contralateral to the neuropathic paw. Nerve injury was performed using the CCI and the SNI models (one group per model) in two separate groups of rats (n = 6) after complete recovery from the surgery for DLF + ALC lesions.
to either the ipsilateral or contralateral DLF did not abolish the neuropathic manifestations and that allodynia and hyperalgesia developed normally in rats sustaining chronic DLF or ALC lesions prior to the induction of neuropathy. Therefore, our results do not support the hypothesis of descending facilitation. In addition, the observed recurrence of neuropathic manifestations appears to be more in line with the original concept of descending inhibitory influences on nociception from brainstem centers (Basbaum and Fields, 1978, 1984). Moreover, other studies reported that neuropathy can activate inhibitory mechanisms of nociception descending in the DLF system (Wall et al., 1988; Sotgiu, 1993; Danziger et al., 2001; Ma and Eisenach, 2003). Apart from some differential effects, our results have revealed that all spinal tract lesions can attenuate tactile allodynia and thermal hyperalgesia. These findings provide clear evidence against the concept of modality-specific segregation of the spinal pathways involved in rostral modulation or signalling of neuropathic manifestations (Bester et al., 2000; Blomqvist and Craig, 2000), since lesion of each tract was able to produce reversible attenuation of all neuropathic manifestations and that these manifestations showed regular development in rats sustaining various spinal tract lesions prior to the induction of mononeuropathies. The observed recurrence of the neuropathic manifestations complements the idea
of plasticity of channeling of nociception through the available pathway. This hypothesis correlates well with clinical observations reporting that under pathological conditions, nociceptive signalling can travel through the DC system (Triggs and Beric, 1994; Beric, 1997). It can be also correlated with clinical observations reporting the failure of cordotomies in relieving chronic pains (Nathan and Smith, 1979; Gybels and Tasker, 1999). Our results showed consistently reversible attenuation of the neuropathic manifestations which reflects a transient interruption of either facilitatory or inhibitory supraspinal control mechanisms. This assumption is supported by previous reports from our laboratory showing short- and long-term modulation of neuropathic-like behavior by interfering with the normal function of the ventral orbital area (Baliki et al., 2003; Al-Amin et al., 2004) or the somatosensory thalamic nuclei (Saade´ et al., 2006). However, there remains the need to explain the following two observations: Why were all the observed effects involving only attenuation of the neuropathic manifestations? Why was attenuation reversible and followed by a full recurrence of these manifestations? Unlike the autotomy model (Wall et al., 1988; Saade´ et al., 1990) spinal lesions (DLF mainly) did not increase the neuropathic manifestations, perhaps, because these manifestations are already set at their maximal level and the lack of attenuation can be
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considered as equivalent to facilitation. The recovery of manifestations following each lesion may indicate a delayed adjustment to the loss of supraspinal influences. A final question that emanates from this discussion: are these manifestations simple spinal reflexes or elements of a more elaborate behavior related to chronic pain? A possible answer is that each of these manifestations depends on a spinal circuitry, but this circuitry is part of spinal and supraspinal network involved in the processing of nociceptive information and the organization of pain-related behavior. Finally, the results of the present study lead to the following conclusions. First, the observed tactile and cold allodynia and heat hyperalgesia are not simple spinal reflexes, but depend to a large extent on supraspinal influences. Second, the reversible attenuation of neuropathic manifestations, produced by selective lesions of the various spinal tracts, provides evidence about the importance of the supraspinal modulatory mechanisms and on the flexibility of the neural network involved in this process. Third, the observed involvement of all the spinal tracts in the modulation of the allodynia and thermal hyperalgesia provides evidence against the concept of specific fiber tracts involved in the processing of the different aspects of neuropathic manifestations.
Acknowledgements The authors thank Sawsan Sharrouf, Bilal Mazeh, Bassem Najm, Nadine Fawaz, and Riad Maalouf for their technical assistance. This project was supported by grant from the Lebanese National Council for Scientific Research.
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