Nerve constriction in the rat: model of neuropathic,... : PAIN (original) (raw)
1. Introduction
Clinically, many different nerve injury or neuropathic conditions exist. Some are diagnosed as causalgia, postherpetic neuralgia or reflex sympathetic dystrophy to mention only a few. In some cases, patients afflicted with one of these neuropathic syndromes express continuous intense pain which is exacerbated by stimuli that are normally innocuous such as light touch (Price et al., 1989; Gracely et al., 1992). Recently, animal models have been developed in an attempt to mimic the effects of nerve injury clinically and thus provide a better understanding of the physiological mechanisms involved in producing and maintaining neuropathic pain. We undertook to expand our studies on the chemical basis of synaptic transmission in spinal sensory mechanisms in normal animals to a nerve constriction model of neuropathic pain. However, during the selection process of a suitable model a number of questions were encountered which were not satisfactorily answered in the literature. These centred mainly on the optimal time to do testing after the nerve constriction was applied and what was appropriate to use as a control.
The first widely accepted animal model of neuropathy was developed by Bennett and Xie (1988), in which loose ligatures were placed around a sciatic nerve in the rat. This rendered an ipsilateral increase in sensitivity to noxious and innocuous mechanical stimuli and a favouring of the respective paw. Since that report, three additional principal models have been reported, by Seltzer et al. (1990), in which only part of the sciatic nerve is tightly ligated, by Kim and Chung (1992) in which tight ligatures are placed around the L5 and 6 spinal nerves, and by Mosconi and Kruger in which short cuffs of polyethylene tubing are inserted around a sensory nerve (Mosconi and Kruger, 1996). This most recent model provides presumably little if any variation in the magnitude of constriction of the sensory nerve between rats of the same size and weight. As our long-term studies are to involve both reflex testing in one project and electrophysiological recording in another, it was important to minimise variation in the degree of nerve constriction and the Mosconi and Kruger model was considered appropriate to accomplish this.
In this study, a 2 mm polyethylene cuff was used to constrict the left common sciatic nerve and the effect of this on the withdrawal threshold of the ipsilateral hind paw was examined using von Frey hairs. Specifically, we determined changes in the withdrawal threshold from the presurgery baseline level and the time course of any such changes. To establish the appropriate control, withdrawal thresholds were also measured in the contralateral hind limb, in both hind limbs of sham-operated rats as well as in both hind limbs of unoperated rats.
2. Methods
2.1 Animals
Experiments were done using adult, male Sprague–Dawley rats (375–425 g) from Harlan Sprague–Dawley, Inc. (Indianapolis, Indiana). They were housed in plastic cages containing wood chip bedding (Hardwood Laboratory Bedding, Northeastern Products Corp., Warrensburg,NY) and maintained on a 12:12 h light:dark cycle (lights on at 07:00 h) with access to food and water ad libitum. Experiments were conducted during the light component of the cycle. Only two rats from the same testing group (i.e. unoperated, sham-operated or cuff-implanted) were together in any cage. Guidelines in The Care and Use of Experimental Animals by the Canadian Council on Animal Care (Vols. I and II) were strictly followed. All experiments were approved by the McGill University Animal Care Committee.
2.2 Surgical procedure
All surgery was done under aseptic conditions. Under Na-pentobarbital anesthesia (50 mg/kg, i.p., Abbott Laboratories, Limited, Dorval, Montreal, Quebec), the left common sciatic nerve (_n_=8) was exposed via blunt dissection through the biceps femoris muscle. The nerve was isolated from surrounding connective tissue using glass probes. Approximately 4–6 mm of the nerve was elevated minimally and held in place using a sterilised glass probe in order to place on the nerve a 2 mm section of split PE-90 polyethylene tubing (Intramedic PE-90, Fisher Scientific Ltd., Montreal, Quebec). The nerve was kept moist using sterile saline at 37.5°C throughout exposure, which occurred approximately for 10 s. The muscle layer was closed using 3-O silk suture thread (Ethicon Inc., Montreal, Quebec) and the shaved skin layer was closed using three stainless steel suture clips (Fine Science Tools, Inc., North Vancouver, British Columbia). Nitrofurazone ointment 0.2% (Univet Pharmaceuticals Ltd., Milton, Ontario) was placed on the skin suture to control any infection and rats were then allowed to recover for 24 h before testing. Sham-operated rats (_n_=8) underwent the same surgical procedure as described above but without implantation of the cuff. During the post-operative period the animals were monitored several times a day. Particular attention was paid to general behaviour and appearance. Body weight was measured once per day.
To examine any effect of Na-pentobarbital anesthesia on responses of rats on the paw withdrawal threshold, 4 of the 8 unoperated control rats were anaesthetised as described above, at the time of surgery of the cuff-implanted and the sham-operated rats. The remaining 4 untreated rats were not taken from their cages until testing was begun.
2.3 Measurement of mechanical hind paw withdrawal threshold
The hind paw withdrawal threshold was determined using von Frey hairs and was expressed in grams. Ten hairs ranging from 0.23 to 59.0 g were used. The value of each hair was confirmed weekly by measuring the magnitude in grams exerted by the hair when applied to a Mettler AE 100 electronic balance. This was done because it was determined that slight fluctuation in the value of a hair may occur after use. If this was the case for a particular hair, the new value in grams, determined using the electronic balance, was used in determining the paw withdrawal threshold.
Application of the von Frey hairs was done using a platform designed and constructed specifically for von Frey hair testing (Pitcher et al., 1999). Briefly described, the platform was made of plexiglass 3 mm thick. It was slightly opaque in appearance and contained 1.5 mm diameter holes in perpendicular rows, 5 mm apart throughout the entire area of the platform. For testing, a rat was placed on this platform which was fixed in a transparent plexiglass observation chamber (30×30×30 cm).
Testing was blind such that the experimenter was not aware of the kind of rat being tested, i.e. unoperated, sham-operated or cuff-implanted. The protocol used in this study was a variation of that described by Takaishi et al. (1996). A testing session for a particular rat began after 5 min of habituation or as soon as the rat stopped exploring and appeared acclimatised to the testing environment. The series of von Frey hairs was applied from below the platform to the plantar surface of the left hind paw in ascending order beginning with the lowest hair (0.23 g). Application was to the central region of the plantar surface avoiding the foot pads. A particular hair was applied until buckling of the hair occurred. This was maintained for approximately 2 s. The hair was applied only when the rat was stationary and standing on all four paws. A withdrawal response was considered valid only if the hind paw was completely removed from the platform. Although infrequent, if a rat walked immediately after application of a hair instead of simply lifting the paw, the hair was reapplied. On rare occasions, the hind paw only flinched after a single application; as the hind paw was not lifted from the platform, this was not considered a withdrawal response.
A trial consisted of application of a von Frey hair to the hind paw five times at 5 s intervals or as soon as the hind paw was placed appropriately on the platform after 5 s. If withdrawal did not occur during five applications of a particular hair, the next larger hair in the series was applied in a similar manner. When the hind paw was withdrawn from a particular hair either four or five times out of the five applications, the value of that hair in grams was considered to be the withdrawal threshold.
Once the threshold was determined for the left hind paw, the same testing procedure was repeated on the right hind paw after 5 min. Second and third testing trials were run for the left and right hind paws, respectively. If the withdrawal threshold in the second or third trial did not match the withdrawal threshold of the previous testing trial(s) in a given hind paw, the next larger hair in the series was tested. This was done until the withdrawal thresholds in three successive trials matched. Only hind paw withdrawal thresholds that remained consistent in the second and third successive trials in unoperated, sham-operated or cuff-implanted rats were used in the data analysis. The total testing time for each rat usually lasted 35 to 40 min.
The baseline withdrawal thresholds of each of the hind paws to von Frey hair application were determined for each rat prior to surgical manipulation (day 0). Testing commenced the following day (day 1). Subsequent testing occurred each day for 2 weeks, then every 2 to 3 days until day 32, every 5 days until day 47, every 7 days until day 61 and then every 14 days until day 145, the last day of testing.
2.4 Statistical analysis
Hind paw withdrawal thresholds were analysed using Kruskal-Wallis one way ANOVA on ranks. Student-Newman-Keuls test was used for post-hoc comparisons between or within groups of animals following ANOVA. Hind paw withdrawal threshold values between different groups of rats or within the same group at different time points were considered significantly different with a P value <0.05.
3. Results
3.1 Effects of cuff-implantation or sham surgery on rat behaviour
Before surgery, upon being placed in the testing chamber, rats from the three groups engaged in general exploration. However, habituation occurred always before or at most shortly after the end of the prescribed 5 min acclimatisation time period. During testing vocalisation did not occur in response to application of any of the von Frey hairs nor was autotomy observed at any time in rats in any of the three groups. Furthermore, throughout the study, rats in each of the groups were well groomed. General appearance, weight as well as the stools were normal throughout the study. However, during approximately the second and third weeks of testing, the claws of the cuff-implanted hind paw were curved and noticeably longer than normal, which is consistent with previous reports (Bennett and Xie, 1988). In some of the cuff-implanted rats, elongation of the claws was observed bilaterally.
Behavioral differences between the unoperated, sham-operated and cuff-implanted rats were also apparent. For example, between days 1 and 30 the cuff-implanted rats spontaneously lifted the ipsilateral hind paw in their housing cages as well as in the testing chamber. ‘Teeth-chattering’ also occurred during this time in these rats. Spontaneous lifting of the injured hind paw was less frequently displayed in sham-operated rats and not at all in unoperated rats. Furthermore, after surgery the cuff-implanted rats held up the ipsilateral hind limb longer and higher while walking; this was not observed in rats in the unoperated or the sham-operated groups. In fact, while walking, the cuff-implanted animals often hopped on the contralateral limb. This was particularly noticeable during the first 2 weeks of the study. Application of even the low threshold von Frey hairs to the hind paws in this group also evoked abrupt paw withdrawal, with the paw remaining elevated for 10 to 30 s in some cases. This was often followed by licking and shaking of the stimulated paw which was not observed in the unoperated nor in the sham-operated rats. Interestingly, common to all of the cuff-implanted rats was their tendency to curve or ‘cup’ the ipsilateral hind paw. Some of these rats even demonstrated ‘ventralflexion’ of the toes of the ipsilateral hind paw and developed calloused skin on the heel and side of the paw which have also been reported in other models of neuropathy in the rat (Kim and Chung, 1992; Na et al., 1996).
Postmortem examination of each of the rats in the cuff-implanted group showed that each implanted cuff remained constricting the common sciatic nerve. It is important to note that there was fibrous tissue development on the sciatic nerve extending 2 to 3 mm on each side of the cuff. In a few of the sham-operated rats, examination revealed some fibrous tissue. However, this was substantially less than that observed in the cuff-implanted rats.
3.2 Baseline hind paw withdrawal threshold in unoperated, sham-operated and cuff-implanted rats
Fig. 1A shows that on day 0, before cuff implantation or sham surgery, the hind paw withdrawal threshold in rats in the unoperated (44.63±6.39 and 43.63±5.56 g for the left and right hind paws, respectively), sham-operated (51.88±4.30 g for each of the hind paws) and cuff-implanted (53.63±4.46 g for each of the hind paws) groups were not statistically different.
Effect of cuff implantation, sham surgery or no surgery on the withdrawal threshold of the left and intact contralateral hind paws in rats. The horizontal axis represents time in days and the vertical axis represents the mean (±SEM) paw withdrawal threshold expressed in grams and determined via application of von Frey hairs. (A) Initially, the mean hind paw withdrawal thresholds in each of the three groups of rats were measured over 15 consecutive days. On day 0, the baseline left and right hind paw withdrawal thresholds in the unoperated, sham-operated and cuff-implanted groups of rats were statistically similar. Cuff implantation and sham surgery were done after baseline testing. In unoperated rats, the mean withdrawal thresholds of both hind paws remained at baseline. However, in the sham-operated group of rats, the mean withdrawal thresholds of the operated paw and the intact contralateral paw decreased. In cuff-implanted rats, the mean paw withdrawal threshold of the nerve-injured hind paw decreased and was lower than withdrawal thresholds in the sham-operated group. Contralateral hind paw withdrawal thresholds were similar to that in the sham-operated group. (B) Between days 16 and 54, rats were tested every 2, 3, 5 and then 7 days. Mean hind paw withdrawal thresholds in the unoperated group of rats remained at baseline and the sham-operated rats remained approximately at 30 g until the onset of recovery between days 42 and 47. In cuff-implanted rats, onset of recovery of the withdrawal threshold occurred between days 23 and 26 while the withdrawal threshold of the contralateral hind paw remained predominantly unchanged.(C) Rats were tested every 14 days between days 61 and 145, the last day of testing. Mean left and right hind paw withdrawal thresholds remained at baseline in the unoperated and in the sham-operated groups of rats. Onset of recovery of the intact hind paw contralateral to the nerve constriction occurred between days 60 and 77. During recovery, hind paw withdrawal thresholds were statistically similar. (* P<0.05 vs. unoperated group, + P<0.05 vs. sham-operated group and •P<0.05 vs. contralateral hind paw; SEM bars directed upward on hollow symbols and downward on solid symbols).
3.3 Unoperated rats
The withdrawal thresholds of each of the hind paws obtained from the 4 rats that were given Na-pentobarbital on day 0 were not significantly different compared to the paw withdrawal thresholds obtained from the 4 unoperated rats that did not receive the anaesthetic. Fig. 1A–C show that left and right hind paw withdrawal threshold values of the 8 rats as one group were not statistically different at any of the testing days compared to the baseline paw withdrawal threshold at day 0.
3.4 Sham-operated rats
On day 1, the mean withdrawal threshold of the left hind paw i.e. ipsilateral to the surgery, was at baseline. However, by day 3 it had decreased to 28.51±6.31 g (P<0.05 vs. left hind paw withdrawal threshold of the unoperated group) and remained approximately at this level up to day 42 (26.96±5.03 g, P<0.05 vs. unoperated). Between days 9 and 23, the mean threshold showed some fluctuation possibly due to day to day handling of the rats. However, between days 47 and 145 the withdrawal thresholds were not significantly from those of the unoperated group (see Fig. 1B,C).
The mean withdrawal threshold of the right hind paw, i.e. contralateral to the surgery, decreased to 30.45±4.85 g on day 1 (P<0.05 vs. right hind paw withdrawal threshold of the unoperated group) and to 14.48±2.21 g on day 3 (P<0.05 vs. unoperated). Fig. 1A,B show that by day 5 the mean withdrawal threshold was 31.06±7.16 g and remained approximately at this level up to day 37 (28.68±5.74 g, P<0.05 vs. unoperated). From day 42 to the end of the study, the right hind paw withdrawal thresholds of the sham-operated and the unoperated groups were not significantly different (see Fig. 1B,C).
3.5 Cuff-implanted rats
The mean withdrawal threshold of the left hind paw, i.e. ipsilateral to the cuff, decreased shortly after surgery (see Fig. 1A). For example, it was 30.38±8.58 g on day 1 (P<0.05 vs. hind paw withdrawal thresholds of the unoperated and the sham-operated groups), 5.51±1.57 g on day 3 (P<0.05 vs. unoperated, sham-operated and the contralateral), 2.95±0.77 g on day 5 (P<0.05 vs. unoperated, sham-operated and contralateral), and remained approximately at this level up to day 23 (3.78±0.45 g, P<0.05 vs. unoperated, sham-operated and contralateral). Fig. 1B,C show that from day 26 (6.15±1.36 g, P<0.05 vs. unoperated, sham-operated and contralateral) to day 145 (26.71±5.67 g, P<0.05 vs. unoperated and sham-operated) the mean threshold returned gradually towards pre-cuff values.
Fig. 1A shows that the right hind paw withdrawal threshold became different from that of the unoperated group only on day 6 (23.35±5.62 g, P<0.05). On day 10 the mean withdrawal threshold was 15.21±2.38 g (P<0.05 vs. unoperated) and remained approximately at this level until day 61 (15.58±2.43 g, P<0.05 vs. unoperated and sham-operated). Fig. 1C shows that between days 60 and 75, the mean threshold began recovering gradually and that on day 145 the mean right hind paw withdrawal threshold had reached 32.16±5.49 g (P<0.05 vs. unoperated). Between days 37 and 145, Fig. 1B,C show that the withdrawal thresholds of each of the hind paws were not significantly different.
4. Discussion
It is demonstrated in this study that unilateral constriction of the left common sciatic nerve, using a variation of the Mosconi and Kruger (1996) technique, gives rise to a marked increase in sensitivity to normally innocuous tactile stimuli in both the nerve-injured as well as the intact contralateral hind paws. Although less in magnitude and duration, surgery alone without nerve constriction also produces a decrease in withdrawal threshold of each of the hind paws. As unoperated rats showed no change from the normal hind paw withdrawal threshold throughout the study, we interpret our findings to suggest that nerve constriction and even the effects of surgery alone establish sustained modifications in sensory processing which maintain long-lasting tactile allodynia ipsi- and contralateral to the nerve constriction or surgical injury.
4.1 No effect of testing protocol on hind paw withdrawal
In unoperated rats the withdrawal threshold of each of the hind paws to the effects of application of von Frey hairs remained unchanged throughout the entire time course of the study. This is interpreted to suggest that a number of important aspects of our testing protocol do not have adverse effects on the paw withdrawal reflex. For example, three tests per hind paw per session yielded the same threshold. Secondly, there was no variation in the thresholds measured once a day every 1, 2, 3, 5, 7 or 14 days which demonstrates that the period between testing sessions also has no effect on the paw withdrawal threshold. Thirdly, there was no change in the threshold over 145 days which reveals no effect of duration of testing on threshold. Thus, the period between testing rats, the duration of testing and the experimental set-up including the surface on which the rats stand while being tested with von Frey hairs provide measurement of stable paw withdrawal thresholds.
4.2 Hind paw withdrawal threshold ipsilateral to cuff implantation
The present study shows that constriction of the common sciatic nerve using a 2 mm polyethylene cuff produces a partial and long-lasting hypersensitivity to normally innocuous tactile stimuli. The onset of this effect occurs as soon as 1 day after cuff implantation and is sustained for at least 145 days with maximal hypersensitivity, measured at 1 to 2 g using von Frey hairs, occurring between 4 and 27 days. Although the cuff-implanted rats appeared well groomed and showed no autotomy or vocalisation during testing, there was a persistent favouring and abrupt lifting and licking of the ipsilateral paw in response to application of von Frey hairs during the first 3 to 4 weeks of testing. Therefore, constriction of the sciatic nerve produces changes in sensory processing which are expressed as decreased withdrawal thresholds to normally innocuous stimuli and as nociceptive behaviour such as paw favouring.
Increased sensitivity of the nerve-injured hind paw in the rat to von Frey hair application is observed in numerous other studies including those using the Bennett and Xie (1988) (Cui et al., 1996, 1997; Ramer et al., 1997; Ramer and Bisby, 1997; Sawin et al., 1997), the Seltzer et al. (1990) (Meyerson et al., 1995; Ren et al., 1996; Stiller et al., 1996; Kim et al., 1997) and the Kim and Chung (1992) (Kim and Chung, 1991; Chung et al., 1993, 1996, 1997; Sheen and Chung, 1993; Qian et al., 1996; Lee and Chung, 1996; Na et al., 1996; Yoon et al., 1996) models of neuropathic pain. Some differences exist though. For example, using the Bennett and Xie technique (Bennett and Xie, 1988) to induce nerve injury, a maximum decrease in the withdrawal threshold to 5–10 g was reported and this decrease persisted up to the end of the study at 28 days (Ramer and Bisby, 1997). Using both the Bennett and Xie (1988) and the Seltzer et al. (1990) techniques, it is reported that an increase in sensitivity of the nerve-injured hind paw to repetitive application of an 0.8 g von Frey hair occurs beginning at least one day after surgery and this effect persists for approximately 28 days with full recovery by 84 days (Kim et al., 1997). Using their own model, Seltzer et al. (1990) report a decrease in withdrawal threshold of the nerve injured paw to approximately 2 g as soon as 1 h after surgery and this effect persists up to the end of the study at 54 days. Also in the Seltzer model, a decrease in the withdrawal threshold to approximately 5 g is reported at 7 and 112 days after surgery (Takaishi et al., 1996). In studies in which the Kim and Chung model of neuropathic pain is used, the onset of the decrease in paw withdrawal threshold to repetitive application of an 0.8 g von Frey hair is reported to occur as soon as 1 (Chung et al., 1997) to 3 (Sheen and Chung, 1993) days after surgery and persist for up to 56 (Chung et al., 1996; Na et al., 1996), 84 (Kim and Chung, 1992) or 140 (Kim et al., 1997) days after surgery.
Therefore, peripheral nerve injury induces a decrease in paw withdrawal threshold which is early in onset and persists for at least several weeks. The differences in magnitude and time course of the effects of nerve injury in the different studies as well as in our own are postulated to be due, at least in part, to the effects of the different kinds of nerve injury techniques on sensory input processing. In fact, in the chronic constriction injury model, there is suggestion that resorption of the chromic gut, which is used for ligatures in some studies, allows the process of recovery to occur (Coggeshall et al., 1993). In addition, there is accumulating evidence that the testing surface, specifically wire mesh, on which rats are tested with von Frey hairs, may produce discomfort for the rat (Kauppila et al., 1998; Mizisin et al., 1998). While many studies report using wire mesh to apply the von Frey hairs, very few indicate the dimensions of the mesh. Therefore, differences in the kinds of testing surfaces used may be another source of inconsistency between studies.
In the present study, the long-lasting tactile hypersensitivity in the cuff-implanted hind paw is considered an appropriate model of neuropathic pain and may be representative of the clinical effects of neuropathy (Price et al., 1989; Gracely et al., 1992).
4.3 Hind paw withdrawal threshold contralateral to cuff implantation
The decrease in the withdrawal threshold of the intact contralateral hind paw was gradual with the maximum mechanical sensitivity occurring approximately 37 days after surgery. It was not considered prior to this time because the sustained decrease in the withdrawal threshold before day 37 was statistically similar to the sham surgery-induced decrease in the contralateral hind paw withdrawal threshold. Therefore, prior to day 37, although we cannot entirely exclude any influence of nerve constriction on the withdrawal threshold of the hind paw contralateral to nerve constriction, such an effect appears to be minimal during this time and is likely predominantly expressed between days 32 and 77 after cuff-implantation.
The bilateral decrease in the hind paw withdrawal threshold shown here is distinct from the almost invariable unilateral decrease reported in most studies to date. Seltzer et al. reported a decrease in the withdrawal threshold of the contralateral hind paw beginning 1 h after surgery at the same von Frey hair threshold as that of the ipsilateral nerve-injured hind paw and this decrease persisted for at least 54 days (Seltzer et al., 1990). Takaishi et al. reported also a contralateral decrease in the threshold of hind paw withdrawal at 1 and 16 weeks after nerve injury (Takaishi et al., 1996). In one study, peripheral nerve constriction is also reported to decrease the hind paw withdrawal latency in the Randall Selitto test (Yu et al., 1996). Bilateral tactile hyperesthesia is also reported following peripheral nerve cryoneurolysis (DeLeo et al., 1994; Willenbring et al., 1994), carrageenan injection into one hind paw (Kissin et al., 1998), transection of the ventral ramus of the spinal nerve L5 (Blenk et al., 1997), and bilateral autotomy has been observed following dorsal rhizotomy (Lombard et al., 1979). Moreover, clinically, the pain associated with causalgia in humans is sometimes found to be manifested opposite to that of the nerve injury (Kozin et al., 1976; Procacci and Maresca, 1987). Thus, in addition to changes in sensory processing ipsilateral to nerve constriction, our data endorse the notion that the physiological mechanisms governing sensory input via the intact contralateral hind paw are also subject to modulation.
Although, at present, we have no comprehensive answer to account for the contralateral effect observed in the present study, several potential explanations may provide some insight to how contralateral sensory input may be modified such that normally innocuous stimuli become painful. At present, both central and peripheral mechanisms must be considered. Perhaps one of the first indications that ipsilateral sensory input may influence sensory processing contralaterally was from Culberson et al. and Light and Perl who reported that the central terminals of primary afferent nerve fibers may project to the contralateral dorsal horn (Culberson et al., 1979; Light and Perl, 1979). However, intriguingly, in the cat and in the opossum these projections were rarely seen in the lumbosacral level of the spinal cord compared to the cervical/brachial levels. More recently, bilateral expression of ‘dark neurons’, presumably the result of transynaptic degeneration subsequent to unilateral nerve constriction, is shown in the superficial laminae of the lumbar spinal dorsal horn (Sugimoto et al., 1990; Hama et al., 1994, 1996). Interestingly, GABAergic neurons which are normally found in the superficial laminae in the lumbar dorsal horn under normal conditions become decreased significantly ipsi- and contralaterally following peripheral nerve constriction (Ibuki et al., 1997). However, whether decreased inhibitory mechanisms centrally contribute to nociceptive behaviour and a decreased withdrawal threshold bilaterally remains to be clarified. Persistent noxious thermal stimulation as well as the tonic effects of formalin injection are also reported to produce bilaterally in the spinal dorsal horn significant increases in membrane-associated protein kinase C, as assayed by quantitative autoradiography of the specific binding of [3H]phorbol-12,13-dibutyrate (Yashpal et al., 1995). Furthermore, unilateral formalin injection is also reported to increase bilaterally in the spinal dorsal horn increased metabolic activity measured by [14C]2-deoxyglucose uptake (Porro et al., 1991; Aloisi et al., 1993). Constriction injury of the sciatic nerve is also shown to increase bilaterally both [3H]phorbol-12,13-dibutyrate binding and [14C]2-deoxyglucose uptake in the spinal dorsal horn (Mao et al., 1993). Surprisingly, constriction of the rat sciatic nerve is also reported to induce a vasodilator response in the contralateral hind paw (Kurvers et al., 1996) which is also referred to as ‘reflex neurogenic inflammation’ and is presumably mediated via connections across the spinal cord (Levine et al., 1985). Furthermore, unilateral nerve injury depresses mRNA levels of a specific Na+ channel subunit, SCN10A, in the ipsilateral as well as the contralateral rat dorsal root ganglia (Scadding, 1994) but increases neuropeptide Y-like immunoreactivity (Rydh-Rinder et al., 1996). Unilateral Freund's complete adjuvant induces an ipsilateral and a delayed contralateral ankle arthritis and bilateral increases in preprotachykinin and calcitonin gene-related peptide in dorsal root ganglia (Donaldson et al., 1995). How these contralateral changes, peripheral and central, come about and influence sensory input is not yet evident. Nevertheless, the concept is put forward here that sensory processing contralateral to nerve injury is manifested via the effects of altered central and perhaps peripheral sensory mechanisms.
The data in this study show that unilateral nerve injury evokes an onset of decreased paw withdrawal threshold followed by a plateau phase and then recovery of the withdrawal thresholds in each of the hind paws. Given that the onset of the contralateral effect follows that of the ipsilateral effect and given also that the commencement of the recovery of the contralateral effect is subsequent to that of the ipsilateral recovery (50 to 55 days later), it is conceivable that altered contralateral modulation of sensory input develops over time and may be mediated primarily by altered sensory processing occurring ipsilaterally. In other words, ipsilateral changes in sensory processing may render contralateral sensory mechanisms sensitive to modulation, the consequence of which results in a decrease in the threshold to the effects of innocuous tactile stimuli. This effect is considerable as the withdrawal threshold of the nerve-constricted and the contralateral hind paw are similarly decreased in the mid and end portions of the study. Thus, a model of central pain is proposed here in which persistent nociceptive input elicits changes in sensory processing at a central level, perhaps bilaterally such that the effects of ipsi- and even contralateral sensory input are altered.
Although bilateral effects of nerve injury as yet appear to be relatively uncommon, it must be considered that a contralateral effect of unilateral nerve injury may potentially dispute any rationale for sham surgery or ‘control’ testing done on the intact limb contralateral to the nerve-injured paw in the experimental animal. Furthermore, careful interpretation is particularly critical of data obtained from nociceptive tests which incorporate bilateral hind paw stimulation such as the hot plate or cold water tests in which both hind paws are simultaneously stimulated. For example, nociceptive behaviour in these cases may be a manifestation of abnormal bilateral nociceptive input rather than of the nerve injured hind paw only.
4.4 Hind paw withdrawal threshold ipsi- and contralateral to sham operation
The main reason for running a group of rats which received only sham surgery without cuff implantation was to determine whether surgical manipulation without nerve constriction is an appropriate control group for comparison to the cuff-implanted rats. Specifically, we are inquiring whether any effect(s) of surgery alone has the capacity to alter the processing of sensory information such that normally unperceived innocuous tactile stimuli evoke a paw withdrawal reflex. It is demonstrated in this study that sham surgery is sufficient to produce a relatively long-lasting increase in sensitivity of the hind paws to the effects of application of von Frey hairs. However, the magnitude is less and the duration is not as long-lasting as that observed in the cuff-implanted rats. Interestingly, the intact contralateral hind paw was also sensitive to the effects of application of von Frey hairs and this was identical to that of the ipsilateral hind paw. Therefore, the effects of surgery are adequate in altering mechanisms of sensory processing.
Surprisingly, cutaneous and muscular incision of the hind limb is generally reported to be devoid of effect on the paw withdrawal threshold. However, both Seltzer et al. (1990) and Takaishi et al. (1996), using the Seltzer et al. model of neuropathic pain demonstrate a bilateral decrease persisting for several days in a substantial number of rats revealed by the decreased mean and the large standard error of the mean in the paw withdrawal threshold in the sham-operated group. Kim and Chung and Blenk et al. report also a decrease in the withdrawal threshold of the sham-operated hind limb (Kim and Chung, 1992; Blenk et al., 1997). However, unequivocal evidence that surgical manipulation without direct nerve injury can influence sensory mechanisms comes from Brennan et al. who reported that surgical incision of the rat foot induces a reliable and quantifiable mechanical allodynia lasting for several days after surgery (Brennan et al., 1996, 1997; Zahn and Brennan, 1998; Zahn et al., 1998). As even sites remote from the wound showed persistent mechanical allodynia, this observation demonstrates that sensitisation of sensory mechanisms may be induced by surgery without manipulation of the sensory nerve. Similar observations have also been made clinically. For example, it is reported that surgery may induce ‘spinal sensitisation’ (Lascelles et al., 1995; Wilder-Smith et al., 1996). Thus, the effects of surgery alone appear to have a substantial effect on sensory input perhaps by upregulating or sensitising sensory information processing. Involvement of central mechanisms at least in part in mediating surgery-induced pain is not unreasonable as this concept concurs with the report that surgical procedure that does not include major nerve damage induces transynaptic degeneration in laminae I-III of the spinal dorsal horn (Nachemson and Bennett, 1993). It is presumed that nociceptor-driven excitotoxic insult in the dorsal horn impairs neurons resulting in postoperative pain. For example, intrathecal non-NMDA receptor antagonists are reported to inhibit pain behaviors in a rat model of postoperative pain (Zahn et al., 1998).
It is important to note that in the present study, the decreased hind paw withdrawal thresholds in the sham-operated group were significantly lower than the baseline threshold as well as the threshold from the unoperated group. Therefore, it is suggested that a sham-operated group of rats or sham surgery done to the hind paw contralateral to nerve injury may be inappropriate to use as ‘control’ in which there is expected to be no change from the baseline withdrawal threshold.
If one considers here that sham surgery involved not only exposure of the sciatic nerve but also slight elevation of the nerve for a very short period of time equivalent to that done necessarily in rats in the neuropathic group in order to place the cuff on the sciatic nerve, then the data must also interpreted to suggest that even minimal physical contact even without constriction of the sciatic nerve may also be sufficient to induce hind limb tactile allodynia.
Presently, we have no definitive explanation for the decreased withdrawal threshold of the contralateral hind paw. Nonetheless, a contralateral effect is at least consistent with the increased sensitivity of the hind paw contralateral to cuff implantation. Therefore, possible involvement of surgery-evoked mechanical hyperesthesia in the decreased withdrawal threshold in the cuff-implanted group of rats is not without consideration. However, given that the effect of sham surgery on withdrawal threshold is significantly less in magnitude than the effect of nerve constriction in the cuff-implanted group and that the effects of sham surgery on the paw withdrawal threshold abate several weeks prior to the effects of cuff-induced nerve constriction, the effects of sham surgery are probably less representative of ‘neuropathic’ pain derived from nerve constriction. Rather, the effects of sham surgery observed here are more indicative of surgical sensitisation as contralateral in addition to the ipsilateral allodynia were detected. It is suggested that the effect of sham surgery may be considered representative of postoperative pain.
4.5 Conclusions
In summary, striking evidence is revealed in this study of three types of allodynia in the sciatic nerve-constricted or surgically-injured rat. Each type has a distinct onset, time course, magnitude and recovery. The marked decrease in the withdrawal threshold of the cuff-implanted hind paw may be indicative of neuropathic allodynia characterized by the remarkably long-lasting tactile hypersensitivity accompanied by a combination of nociceptive behaviors. The second type of allodynia was seen in operated but not in unoperated controls and is therefore suggested to be a model of surgical pain. It is also induced unilaterally but expressed bilaterally. The third type of allodynia may be a model central pain and it is demonstrated by the decreased withdrawal threshold of the intact hind paw contralateral to the cuff-implanted paw. Although it is initially less in magnitude compared to that of the neuropathic allodynia, it is slower in onset and later in recovery. Therefore, it is speculated that central pain may be established and maintained via the peripheral and/or central effects of nerve constriction.
Acknowledgements
This study was supported by a grant from the Medical Research Council of Canada to JLH. GMP was supported by Student Fellowships from the Royal Victoria Hospital Research Institute, McGill Faculty of Medicine and the Funds pour la formation de chercheurs et l'aide à la recherche (Province of Quebec).
References
Aloisi AM, Porro CA, Cavazzuti M, Baraldi P, Carli G. Mirror pain in the formalin test: behavioral and 2-deoxyglucose studies. Pain. 1993;55:267-273.
Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 1988;33:87-107.
Blenk KH, Häbler HJ, Jänig W. Neomycin and gadolinium applied to an L5 spinal nerve lesion prevent mechanical allodynia-like behaviour in rats. Pain. 1997;70:155-165.
Brennan TJ, Vandermeulen EP, Gebhart GF. Characterization of a rat model of incisional pain. Pain. 1996;64:493-501.
Brennan TJ, Umali EF, Zahn PK. Comparison of pre- versus post-incision administration of intrathecal bupivacaine and intrathecal morphine in a rat model of postoperative pain. Anaesthesiology. 1997;87:1517-1528.
Chung K, Kim HJ, Na HS, Park MJ, Chung JM. Abnormalities of sympathetic innervation in the area of an injured peripheral nerve in a rat model of neuropathic pain. Neurosci Lett. 1993;162:85-88.
Chung K, Yoon YW, Chung JM. Sprouting sympathetic fibers form synaptic varicosities in the dorsal root ganglion of the rat with neuropathic injury. Brain Res. 1997;751:275-280.
Chung KS, Lee BH, Yoon YW, Chung JM. Sympathetic sprouting in the dorsal root ganglia of the injured peripheral nerve in a rat neuropathic pain model. J Comp Neurol. 1996;376:241-252.
Coggeshall RE, Dougherty PM, Pover CM, Carlton SM. Is large myelinated fiber loss associated with hyperalgesia in a model of experimental peripheral neuropathy in the rat. Pain. 1993;52:233-242.
Cui JG, Linderoth B, Meyerson BA. Effects of spinal cord stimulation on touch-evoked allodynia involve GABAergic mechanisms: An experimental study in the mononeuropathic rat. Pain. 1996;66:287-295.
Cui JG, Sollevi A, Linderoth B, Meyerson BA. Adenosine receptor activation suppresses tactile hypersensitivity and potentiates spinal cord stimulation in mononeuropathic rats. Neurosci Lett. 1997;223:173-176.
Culberson JC, Haines DE, Kimmel DL, Brown PB. Contralateral projection of primary afferent fibers to mammalian spinal cord. Exp Neurol. 1979;64:83-97.
DeLeo JA, Coombs DW, Willenbring S, Colburn RW, Fromm C, Wagner R, Twitchell BB. Characterization of a neuropathic pain model: sciatic cryoneurolysis in the rat. Pain. 1994;56:9-16.
Donaldson LF, McQueen DS, Seckl JR. Neuropeptide gene expression and capsaicinsensitive primary afferents: maintenance and spread of adjuvant arthritis in the rat. J Physiol (Lond.). 1995;486:473-482.
Gracely RH, Lynch SA, Bennett GJ. Painful neuropathy: altered central processing maintained dynamically by peripheral input. Pain. 1992;51:175-194.
Hama AT, Sagen J, Pappas GD. Morphological characterization of dorsal horn spinal neurons in rats with unilateral constriction nerve injury: a preliminary study. Neurol. Res. 1994;16:297-304.
Hama AT, Pappas GD, Sagen J. Adrenal medullary implants reduce transsynaptic degeneration in the spinal cord of rats following chronic constriction nerve injury. Exp Neurol. 1996;137:81-93.
Ibuki T, Hama AT, Wang XT, Pappas GD, Sagen J. Loss of GABA-immunoreactivity in the spinal dorsal horn of rats with peripheral nerve injury and promotion of recovery by adrenal medullary grafts. Neuroscience. 1997;76:845-858.
Kauppila T, Kontinen VK, Pertovaara A. Weight bearing of the limb as a confounding factor in assessment of mechanical allodynia in the rat. Pain. 1998;74:55-59.
Kim KJ, Yoon YW, Chung JM. Comparison of three rodent neuropathic pain models. Exp Brain Res. 1997;113:200-206.
Kim SH, Chung JM. Sympathectomy alleviates mechanical allodynia in an experimental animal model for neuropathy in the rat. Neurosci Lett. 1991;134:131-134.
Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain. 1992;50:355-363.
Kissin I, Lee SS, Bradley EL Jr. Effect of prolonged nerve block on inflammatory hyperalgesia in rats: prevention of late hyperalgesia. Anaesthesiology. 1998;88:224-232.
Kozin F, McCarty DJ, Sims J, Genant H. The reflex sympathetic dystrophy syndrome: clinical and histologic studies: evidence for bilaterality, response to corticosteroids and articular involvement. Am J Med. 1976;60:321-331.
Kurvers HAJM, Tangelder GJ, De Mey JGR, Slaaf DW, Van den Wildenberg FAJM, Kitslaar PJEHM, Reneman RS, Rouwet EV, Jacobs MJHM. Skin blood flow disturbances in the contralateral limb in a peripheral mononeuropathy in the rat. Neuroscience. 1996;74:935-943.
Lascelles BDX, Waterman AE, Cripps PJ, Livingston A, Henderson G. Central sensitisation as a result of surgical pain: investigation of the pre-emptive value of pethidine for ovariohysterectomy in the rat. Pain. 1995;62:201-212.
Lee DH, Chung JM. Neuropathic pain in neonatal rats. Neurosci Lett. 1996;209:140-142.
Levine JD, Dardick SJ, Basbaum AI, Scipio E. Reflex Neurogenic inflammation I: contribution of the peripheral nervous system to spatially remote inflammatory responses that follow injury. J Neurosci. 1985;5:1380-1386.
Light AR, Perl ER. Re-examination of the dorsal root projection to the spinal dorsal horn including observations on the differential termination of coarse and fine fibers. J Comp Neurol. 1979;186:117-132.
Lombard MC, Nashold BS Jr, Albe-Fessard D, Salman N, Sakr C. Deafferentation hypersensitivity in the rat after dorsal rhizotomy: a possible animal model of chronic pain. Pain. 1979;6:163-174.
Mao J, Mayer DJ, Hayes RL, Price DD. Spatial patterns of increased spinal cord membrane-bound protein kinase C and their relation to increases in 14C-2-deoxyglucose metabolic activity in rats with painful peripheral mononeuropathy. J Neurophysiol. 1993;70:470-481.
Meyerson BA, Ren B, Herregodts P, Linderoth B. Spinal cord stimulation in animal models of mononeuropathy: effects on the withdrawal response and the flexor reflex. Pain. 1995;61:229-243.
Mizisin AP, Kalichman MW, Garrett RS, Dines KC. Tactile hyperesthesia, altered epidermal innervation and plantar nerve injury in the hindfeet of rats housed on wire grates. Brain Res. 1998;788:13-19.
Mosconi T, Kruger L. Fixed-diameter polyethylene cuffs applied to the rat sciatic nerve induce a painful neuropathy: ultrastructural morphometric analysis of axonal alterations. Pain. 1996;64:37-57.
Na HS, Yoon YW, Chung JM. Both motor and sensory abnormalities contribute to changes in foot posture in an experimental rat neuropathic model. Pain. 1996;67:173-178.
Nachemson AK, Bennett GJ. Does pain damage spinal cord neurons? Transsynaptic degeneration in rat following a surgical incision. Neurosci Lett. 1993;162:78-80.
Pitcher GM, Ritchie J, Henry JL. Paw withdrawal threshold in the von Frey hair test is influenced by the surface on which the rat stands. J Neurosci Methods. 1999;87:185-193.
Porro CA, Cavazzuti M, Galetti A, Sassatelli L, Barbieri GC. Functional activity mapping of the rat spinal cord during formalin-induced noxious stimulation. Neuroscience. 1991;41:655-665.
Price DD, Bennett GJ, Rafii A. Psychophysical observations on patients with neuropathic pain relieved by a sympathetic block. Pain. 1989;36:273-288.
Procacci P, Maresca M. Reflex sympathetic dystrophies and algodystrophies: historical and pathogenic considerations. Pain. 1987;31:137-146.
Qian J, Brown SD, Carlton SM. Systemic ketamine attenuates nociceptive behaviors in a rat model of peripheral neuropathy. Brain Res. 1996;715:51-62.
Ramer MS, French GD, Bisby MA. Wallerian degeneration is required for both neuropathic pain and sympathetic sprouting into the DRG. Pain. 1997;72:71-78.
Ramer MS, Bisby MA. Rapid sprouting of sympathetic axons in dorsal root ganglia of rats with a chronic constriction injury. Pain. 1997;70:237-244.
Ren B, Linderoth B, Meyerson BA. Effects of spinal cord stimulation on the flexor reflex and involvement of supraspinal mechanisms: an experimental study in mononeuropathic rats. J Neurosurg. 1996;84:244-249.
Rydh-Rinder M, Holmberg K, Elfvin LG, Wiesenfeld-Hallin Z, Hökfelt T. Effects of peripheral axotomy on neuropeptides and nitric oxide synthase in dorsal root ganglia and spinal cord of the guinea pig: an immunohistochemical study. Brain Res. 1996;707:180-188.
Sawin PD, Traynelis VC, Rich G, Smith BA, Maves TJ, Follett KA, Moore SA. Chymopapain-induced reduction of proinflammatory phospholipase A2 activity and amelioration of neuropathic behavioral changes in an in vivo model of acute sciatica. J Neurosurg. 1997;86:998-1006.
Scadding JW., 1994. Peripheral neuropathies. In: Wall PD, Melzack R, editors., Textbook of pain. Churchill Livingstone, New York, p. 667.
Seltzer Z, Dubner R, Shir Y. A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain. 1990;43:205-218.
Sheen K, Chung JM. Signs of neuropathic pain depend on signals from injured nerve fibers in a rat model. Brain Res. 1993;610:62-68.
Stiller CO, Cui JG, O'Connor WT, Brodin E, Meyerson BA, Linderoth B. Release of gammaaminobutyric acid in the dorsal horn and suppression of tactile allodynia by spinal cord stimulation in mononeuropathic rats. Neurosurgery. 1996;39:367-374.
Sugimoto T, Bennett GJ, Kajander KC. Transsynaptic degeneration in the superficial dorsal horn after sciatic nerve injury: effects of a chronic constriction injury, transection, and strychnine. Pain. 1990;42:205-213.
Takaishi K, Eisele JH Jr, Carstens E. Behavioral and electrophysiological assessment of hyperalgesia and changes in dorsal horn responses following partial sciatic nerve ligation in rats. Pain. 1996;66:297-306.
Wilder-Smith OHG, Tassonyi E, Senly C, Otten P, Arendt-Nielsen L. Surgical pain is followed not only by spinal sensitisation but also by supraspinal antinociception. Br J Anaesth. 1996;76:816-821.
Willenbring S, DeLeo JA, Coombs DW. Differential behavioral outcomes in the sciatic cryoneurolysis model of neuropathic pain in rats. Pain. 1994;58:135-140.
Yashpal K, Pitcher GM, Parent A, Quirion R, Coderre TJ. Noxious thermal and chemical stimulation induce increases in 3H-phorbol 12,13-dibutyrate binding in spinal cord dorsal horn as well as persistent pain and hyperalgesia, which is reduced by inhibition of protein kinase C. J Neurosci. 1995;15:3263-3272.
Yoon YW, Na HS, Chung JM. Contributions of injured and intact afferents to neuropathic pain in an experimental rat model. Pain. 1996;64:27-36.
Yu LC, Hansson P, Lundeberg T. The calcitonin generelated peptide antagonist CGRP8-37 increases the latency to withdrawal responses bilaterally in rats with unilateral experimental mononeuropathy, an effect reversed by naloxone. Neuroscience. 1996;71:523-531.
Zahn PK, Umali E, Brennan TJ. Intrathecal non-NMDA excitatory amino acid receptor antagonists inhibit pain behaviors in a rat model of postoperative pain. Pain. 1998;74:213-223.
Zahn PK, Brennan TJ. Lack of effect of intrathecally administered N-methyl-D-aspartate receptor antagonists in a rat model for postoperative pain. Anaesthesiology. 1998;88:143-156.
Keywords:
Allodynia; Neuropathic pain; Central pain; Postoperative pain; Central sensitisation; Rat
© 1999 Lippincott Williams & Wilkins, Inc.