A new model of sciatic inflammatory neuritis (SIN):... : PAIN (original) (raw)
1. Introduction
Immune activation near peripheral nerves may play a greater role in pathological pain than previously thought. Approximately half of neuropathies involve inflammation and/or infection near or within the nerves rather than physical trauma (Bourque et al., 1985; Said and Hontebeyrie-Joskowicz, 1992). Furthermore, immune activation naturally occurs following nerve trauma (Frisen et al., 1993). Thus, immune activation is associated with all neuropathies.
Experiments with rats and Aplysia provide evidence for pain due to immune activation near peripheral nerves. Sensory nerve damage in Aplysia enhances pain responses (Walters, 1994) correlated with recruitment of immunocytes (immune-like cells of Aplysia) (Clatworthy et al., 1994a). Immunocytes function like macrophages, releasing proinflammatory cytokine-like molecules (interleukin-1- (IL1-) like and tumor necrosis factor- (TNF-) like) at the injury site (Clatworthy, 1999). These alter neuronal ion channels, causing hyperexcitability (Sawada et al., 1990; Szucs et al., 1992). Hyperexcitability of injured nerves is greater in the presence of activated immunocytes (Clatworthy and Grose, 1999), and IL1 enhances nerve injury hyperexcitability (Clatworthy et al., 1994b).
In rats, nerve injury causes pain, axonal hyperexcitability, and Wallerian degeneration (Papir-Kricheli and Devor, 1988; Myers et al., 1996a). Both degeneration and pain have been linked to macrophages recruited to the injury (Beuche and Friede, 1984; Myers et al., 1993; Liu et al., 2000b). Delaying macrophage recruitment delays both neuropathic pain and Wallerian degeneration (Myers et al., 1996b; Ramer et al., 1997). In contrast, pain is enhanced by attracting activated immune cells to the injury (Maves et al., 1993; Clatworthy et al., 1995). Moreover, macrophage-derived IL1 and TNF increase at sites of nerve trauma (Leskovar et al., 2000), and blocking IL1 (Sommer et al., 1999) or TNF (Illich et al., 1997; Sommer et al., 1997, 1998) reduces thermal hyperalgesia and mechanical allodynia associated with sciatic nerve injury.
Pain facilitation can occur even in the absence of physical trauma to peripheral nerves. Simply placing gut suture near the sciatic nerve causes hyperalgesia (Maves et al., 1993). This pain has been suggested to result from peri-sciatic immune activation since gut suture activates macrophages (Watkins et al., 1995). In Aplysia, immunologically activating immunocytes near nerves increases excitability (Clatworthy et al., 1994a). Thus immune activation near healthy peripheral nerves appears sufficient to create exaggerated pain states (Watkins et al., 1995). Even killed bacteria applied over rat sciatic nerves induce allodynia and hyperalgesia (Eliav et al., 1999). Such changes are mimicked by proinflammatory cytokines. TNF injected into the sciatic nerve produces thermal hyperalgesia, mechanical allodynia (Wagner and Myers, 1996), endoneurial inflammation, demyelination, and axonal degeneration (Redford et al., 1995). Further, TNF applied to the sciatic nerve induces ectopic activity in single primary afferent nociceptive fibers (Sorkin et al., 1997).
Because of the potential importance of understanding how immune activation near healthy peripheral nerves alters pain processing, we have developed a new rat model for examining sciatic inflammatory neuritis (SIN). The following series of experiments are the first characterization of the pain changes created by localized peri-sciatic immune activation with yeast cell walls (zymosan) and the proinflammatory cytokine high mobility group-1 (HMG).
2. Materials and methods
2.1. General methods
2.1.1. Subjects
Pathogen-free male Sprague Dawley rats (350–450 g; Harlan Labs, Madison, WI, USA) were housed in a light (12:12 light/dark cycle; lights on at 07:00 h) and temperature (23±2°C) controlled room with standard rodent chow and water available ad libitum. Behavioral testing occurred between 07:00 and 13:00 h. All procedures were approved by the Institutional Animal Care and Use Committee and in accordance with IASP guidelines (Zimmermann, 1983).
2.1.2. Drugs
Zymosan (Sigma Chemical Co., St. Louis, MO, USA) was suspended in incomplete Freund's adjuvant (Sigma Chemical Co., St. Louis, MO, USA). HMG was produced as previously described (Wang et al., 1999) and diluted in endotoxin-free physiological saline. Drugs were mixed fresh daily.
2.1.3. Chronic peri-sciatic catheter surgery
Preliminary studies indicated that zymosan applied during surgery induced mechanical allodynia, but only after considerable delay. Allodynia was not observed through 6 h after surgery but robust allodynia was observed at 24 h (Hammack et al., 1999). Since anesthetics alter immune function (Lockwood et al., 1993; Sato, 1995; Miller et al., 1996), this suggested that behavioral changes might be more rapid and/or robust if immune challenge was not compromised by anesthesia. Thus, a chronic indwelling left peri-sciatic catheter system was developed to allow 4–5 days of post-surgical recovery prior to zymosan administration.
Peri-sciatic catheters were constructed from sterile gelfoam (Upjohn, Kalamazoo, MI, USA) aseptically cut into 2.9-cm (L)×5-mm (W)×9-mm (H) strips. One end was bisected (3.5 mm W) to a depth of 1.5 cm to allow a 7-cm sterile silastic tube (Helix Medical Inc., Carpinteria, CA, USA) to be sutured (Ethicon, Somerville, NJ, USA; 4-0 sterile silk) inside.
Isoflurane (Phoenix Pharm., St. Joseph, MO, USA) was chosen as the anesthetic as it has minimal effect on the immune function (Sato, 1995). After aseptic exposure of the left sciatic nerve at mid-thigh level by blunt dissection, the gelfoam was threaded around the nerve so as to minimize nerve displacement. Suturing and insertion of a sterile ‘dummy’ injection tube (PE-50; Becton Dickinson, Sparks, MD, USA) during implantation maintained catheter patency and ensured replicable drug delivery close to the nerve. After anchoring to the muscle, the external end was tunneled subcutaneously to exit 1 cm rostral to the tail base. After removal of the ‘dummy’ injector, the external end of the silastic tube was protected as described previously (Milligan et al., 1999). Less than 1% of rats show any altered sensory or motor effects. These were excluded. All catheters were verified at sacrifice by visual inspection. Data were only analyzed from confirmed sites.
The catheter was used for a single injection 4–5 days after surgery. Injections were conducted in freely moving rats by inserting PE-50 tubing 7.3 cm through the silastic catheter after voiding. Injections were followed by a 20-μl saline flush.
2.1.4. Hargreaves test for thermal hyperalgesia
The Hargreaves test (Hargreaves et al., 1988) was performed as previously described (Milligan et al., 2000, 2001).
2.1.5. von Frey test for mechanical allodynia
The von Frey test (Chaplan et al., 1994) was performed within the sciatic nerve innervation area of the hindpaws as previously described (Milligan et al., 2000, 2001). The behavioral responses were used to calculate the 50% paw withdrawal threshold, by fitting a Gaussian integral psychometric function using a maximum-likelihood fitting method (Harvey, 1986; Treutwein and Strasburger, 1999), as described in detail previously (Milligan et al., 2000, 2001). This allows parametric analyses (Milligan et al., 2000, 2001).
2.1.6. Statistics
Data from the Hargreaves test were analyzed as withdrawal latency. Data from the von Frey test were analyzed as the interpolated 50% threshold in log base 10 of stimulus intensity (monofilament stiffness in grams×104), and tissue weights as milligrams. Pre-drug baseline (BL) measures were compared using one-way analyses of variance (ANOVAs). Post-drug timecourse measures and tissue weights were analyzed by repeated measures ANOVAs followed by Fisher's protected least significant difference post hoc analyses.
2.2. Experiment 1: assessment of the potential effect of a chronic indwelling peri-sciatic catheter on basal mechanical responses
We first assessed whether implantation of the left peri-sciatic catheter (_n_=9) would affect responses to the von Frey test. Responses of both hindpaws were assessed prior to, and again 4–5 days after, catheter implantation.
Additional catheterized rats (_n_=6) were injected with 50 μl Evans blue dye. The gelfoam implant was examined for dye distribution 0.5 h later.
2.3. Experiment 2: effects of peri-sciatic and intra-muscular zymosan on responsivity to low threshold mechanical stimuli and immune organ weight
A range of zymosan doses was tested to determine any altered behavioral responses. We also determined whether close proximity between the zymosan and sciatic nerve was critical for behavioral changes. Here, the catheter was implanted at the same left mid-thigh level in adjoining muscle (biceps femoris); all other aspects of implantation were identical to that used for peri-sciatic placements.
von Frey assessments of all four paws were recorded to determine site specificity. Assessments of hindpaw responses were as described above. Front paw assessments were conducted identically, with the exception that 0.023–3.630 g Semmes–Weinstein monofilaments (von Frey hairs; Stoelting, Wood Dale, IL, USA) were used, based on pilot studies.
Behavioral measures were assessed prior to, and 3 and 24 h after, zymosan or equivolume (50 μl) vehicle. A 6 (0, 4, 40, 160, or 400 μg left peri-sciatic zymosan; or 40 μg zymosan into adjoining muscle)×2 (3 or 24 h)×4 (left and right hindpaws and forepaws) design was used with 7–8 rats/group. Zymosan doses were based on pilot studies.
Upon completion of behavioral testing, tissues were harvested and weighed. These were collected from all groups except 400 μg zymosan, as pilot studies indicated that the unilateral and bilateral phenomena we wished to pursue in future studies were induced by the lower doses. Thus, we wanted to assess the potential spread of zymosan in these lower dose groups. The immune tissues collected (popliteal, iliac, and inguinal lymph nodes and spleen) would enlarge in response to zymosan (Waynforth, 1980); thus, they served as indicators of zymosan spread from the gelfoam.
2.4. Experiment 3: assessment of whether the allodynic effects of peri-sciatic zymosan are restricted to zymosan
To determine whether zymosan is unique in its ability to induce allodynia, a second immune stimulus was examined. Since we have recently begun investigating the effects of HMG (Hansen et al., 2000), a newly recognized putative proinflammatory cytokine (Wang et al., 1999), we tested the effect of HMG (0.5 or 3 μg) vs. equivolume (5 μl) vehicle (_n_=6–7/group). Assessments of von Frey responses were conducted prior to and 0.5, 1, 2, 3, and 24 h after drug injection. Doses and timepoints were based on pilot studies.
2.5. Experiment 4: assessment of the effect of peri-sciatic zymosan on territorial and extra-territorial mechanical thresholds
This experiment examined whether zymosan-induced allodynias are restricted to the sciatic nerve territory, or whether extra-territorial allodynia also occurs in skin innervated by the saphenous nerve. Extra-territorial allodynia would support a central site of sensitization since these nerves do not communicate peripherally (Tal and Bennett, 1994). von Frey responses were recorded from both the sciatic and saphenous hindpaw innervation zones of both the left and right hindpaws both prior to and 24 h after zymosan or vehicle (_n_=6–8/group). Thus, a 5 (0, 4, 40, 160, or 400 μg left peri-sciatic zymosan)×4 (left and right saphenous and sciatic nerves) design was used. Animals were not tested at 3 h after peri-sciatic injections in order to avoid sensitization of behavioral responses due to the number of von Frey trials involved.
2.6. Experiment 5: assessment of the potential effects of peri-sciatic zymosan on thermal responsivity 24 h later
Hindpaw thermal responses were measured prior to and 24 h after peri-sciatic injections. The latter timepoint was chosen since mechanical allodynia was maximal at this time. A 5 (0, 4, 40, 160, or 400 μg zymosan)×2 (left and right hindpaw) design was used with 7–8 rats/group.
2.7. Experiment 6: assessment of the potential effects of peri-sciatic zymosan on thermal responsivity 1, 3, and 24 h later
The present study tested whether thermal hyperalgesia might be evident at shorter time intervals after injection of peri-sciatic zymosan. Thus, hindpaw thermal response thresholds were measured prior to and 1, 3, and 24 h after peri-sciatic injection. A 2 (0 and 40 μg zymosan)×3 (1, 3, and 24 h)×2 (left and right hindpaw) design were used with six–seven rats/group. Only one dose of zymosan was tested to conserve animals. The 40 μg dose was chosen as it is the lowest dose that produces robust bilateral allodynia. In addition, low threshold mechanical thresholds were assessed at the end of Hargreaves testing on days 1 and 2 (approximately 3.5 and 24.5 h after peri-sciatic injection). These additional data were collected to serve as positive controls that the peri-sciatic zymosan injections were behaviorally effective.
3. Results
3.1. Experiment 1: assessment of the potential effect of a chronic indwelling peri-sciatic catheter on basal mechanical responses
The 50% thresholds for responding to von Frey stimuli in intact rats were 4.80±0.07 and 4.88±0.004 for the left and right hindpaws, respectively. When tested again 5 days after implantation, their 50% thresholds were 4.87±0.03 and 4.90±0.04 for the left and right hindpaws, respectively. These pre-surgical and post-surgical von Frey thresholds are not different by ANOVA.
Examination of Evans blue dye distribution in the gelfoams revealed that the dye was localized wholly within the gelfoam and contacted the entire surface of the underlying sciatic nerve.
3.2. Experiment 2: effects of peri-sciatic and intra-muscular zymosan on immune organ weight and responsivity to low threshold mechanical stimuli
Zymosan appeared restricted to the peri-sciatic gelfoam since neither the chain of draining lymph nodes (ipsilateral popliteal, inguinal, and iliac lymph nodes (Waynforth, 1980)) nor distant lymph tissues (contralateral popliteal and inguinal lymph nodes, spleen) enlarged compared to vehicle controls (Fig. 1). ANOVAs revealed that zymosan dose had no significant effect on any tissue measured.
Lack of effect of 4 μg peri-sciatic zymosan (‘B’ bars), 40 μg peri-sciatic zymosan (‘C’ bars), 40 μg intra-muscular zymosan (‘D’ bars), or 160 μg peri-sciatic zymosan (‘E’ bars) to either enlarge lymph nodes or shrink lymph organs, compared to peri-sciatic vehicle (‘A’ bars). Ipsilateral popliteal lymph nodes (Panel (A)), contralateral popliteal lymph nodes (Panel (B)), ipsilateral inguinal nodes (Panel (C)), contralateral inguinal nodes (Panel (D)), iliac nodes (Panel (E)), and spleen (Panel (F)) were collected 24 h after peri-sciatic or intra-muscular injections. If zymosan escaped the gelfoam injection site, enlargement of the sequential chain of lymph nodes that drain the hindleg (popliteal, inguinal, and iliac) would be expected on the ipsilateral side. If zymosan reached the general circulation, enlargement of contralateral lymph nodes and spleen would be expected. Thus, the pattern of lymph node and organ weights supports that zymosan remains restricted to the gelfoam.
Zymosan also appeared to remain restricted to the peri-sciatic gelfoam, rather than reaching the general circulation, given that no dose of zymosan altered mechanical sensitivity of the front paws (Fig. 2). No BL differences were observed. At 3 and 24 h after injection, no differences in mechanical responses were found either comparing drug groups or comparing left and right forepaws.
Lack of effect of 4 μg peri-sciatic zymosan (Panel (B)), 40 μg peri-sciatic zymosan (Panel (C)), 40 μg intra-muscular zymosan (Panel (D)), 160 μg peri-sciatic zymosan (Panel (E)), or 400 μg peri-sciatic zymosan (Panel (F)) on mechanical response thresholds of the forepaws, compared to vehicle controls (Panel (A)). In each panel, data for left forepaws are represented by white squares and for right forepaws by black squares. Behavioral measures were recorded prior to (BL) and 3 and 24 h after injection.
Regarding mechanical sensitivity of the hindpaws, no BL differences were observed. However, after injection, marked differences in mechanical thresholds were observed between groups (Fig. 3). ANOVA revealed main effects of zymosan dose (_F_5,82=58.387, P<0.0001) and laterality (_F_1,82=22.096, P<0.0001), and an interaction between zymosan dose and laterality (_F_5,82=12.763, P<0.0001). Post hoc means comparisons revealed several important points. After 4 μg zymosan (Fig. 3B), mechanical allodynia was observed in the left (ipsilateral) hindpaw compared to the right (contralateral) hindpaw (P<0.0001). Mechanical responses of the right hindpaw after 4 μg zymosan did not differ from that following vehicle (Fig. 3A), supporting that 4 μg zymosan induced only a unilateral allodynia ipsilateral to the site of injection. Bilateral mechanical allodynia was observed after 40, 160, or 400 μg peri-sciatic zymosan (Fig. 3C,E,F). That is, for each of these groups, the thresholds of the left and right hindpaws did not differ, but the thresholds for both the left and right paws for all of these groups were reliably different from those of the vehicle controls (P<0.0001). Importantly, post hoc comparisons revealed no allodynia in either paw following 40 μg zymosan injections into the adjoining muscle (Fig. 3D). That is, left hindpaw thresholds after intra-muscular zymosan did not differ from the left hindpaw responses of vehicle controls, but did reliably differ from the left hindpaw responses of all other groups (P<0.0001). Similarly, the right hindpaw thresholds of the intra-muscular zymosan group did not differ from the right hindpaw responses of either the vehicle controls or the 4 μg zymosan group but did reliably differ from the right paw responses of the 40, 160, and 400 μg zymosan groups (P<0.0001). These intra-muscular zymosan data indicate both that the zymosan is not migrating beyond the gelfoam to create pain changes and that close proximity between the zymosan (and presumably the resultant immune activation) and the sciatic nerve is critical to the effects observed.
Dose-dependent effects of peri-sciatic zymosan on responses to low threshold mechanical stimuli. In each panel, data for left hindpaws are represented by white squares and for right hindpaws by black squares. Responses to low threshold mechanical stimuli were assessed using the von Frey test prior to (BL) and 3 and 24 h after peri-sciatic or intra-muscular injection. (Panel (A)) Peri-sciatic vehicle (incomplete Freund's adjuvant) injection has no effect on von Frey responses of either hindpaw. (Panel (B)) Peri-sciatic 4 μg zymosan produces unilateral allodynia. That is, allodynia is observed in the left hindpaw, ipsilateral to zymosan injection. The right (contralateral) hindpaw shows no change in low threshold mechanical responses. (Panels (C, E, F)) Higher doses of peri-sciatic zymosan (40, 160, and 400 μg) produce bilateral allodynia. That is, allodynia is observed in both the left (ipsilateral) and right (contralateral) hindpaw. (Panel (D)) Failure of 40 μg zymosan to produce allodynia in either hindpaw when administered into gelfoam implanted within adjoining muscle. These results suggest that (a) immune activation must occur in close proximity to peripheral nerves to create allodynia and (b) zymosan spread to systemic circulation cannot explain allodynias created by peri-sciatic zymosan.
3.3. Experiment 3: assessment of whether the allodynic effects of peri-sciatic zymosan are restricted to zymosan
No BL differences were found (Fig. 4). In contrast, ANOVA revealed main effects of peri-sciatic drug (_F_2,34=28.133, P<0.0001), laterality (_F_1,34=22.783, P<0.0001), and time (_F_4,136=3.687, P<0.01), and interactions between peri-sciatic drug and laterality (_F_2,34=5.289, P<0.01) and between time, peri-sciatic dose, and laterality (_F_8,136=3.427, P<0.01). Post hoc comparisons revealed that peri-sciatic injection of 0.5 μg HMG produced decreased mechanical response thresholds of the left hindpaw relative to the right at all times tested (P values <0.01), except at 2 h (Fig. 4B). Similarly, the right hindpaw responses after 0.5 μg HMG did not differ from vehicle, with the one exception of the 2-h timepoint (P<0.025). Thus, 0.5 μg HMG predominantly produced a unilateral allodynia. Peri-sciatic injection of 3 μg HMG produced a bilateral allodynia as both left and right hindpaw responses differed from those of vehicle controls (P values <0.05). Furthermore, response thresholds of the left and right hindpaws after 3 μg peri-sciatic HMG did not reliably differ at any time tested. Thus, zymosan is not unique in its ability to produce allodynia after peri-sciatic administration.
Generality of unilateral and bilateral allodynic effects of peri-sciatically injected immune stimuli. In each panel, data for left hindpaws are represented by white squares and for right hindpaws by black squares. (Panel (A)) Peri-sciatic injection of equivolume (5 μl) vehicle had no reliable effect on von Frey responses measured from the left and right hindpaws. (Panel (B)) Peri-sciatic injection of 0.5 μg HMG produced robust allodynia in the ipsilateral (left) hindpaw. A transient decrease in mechanical response threshold was observed only at 2 h after 0.5 μg HMG injection. (Panel (C)) Peri-sciatic injection of 3 μg HMG produced robust allodynia in both the left and right hindpaws.
3.4. Experiment 4: assessment of the effect of peri-sciatic zymosan on territorial and extra-territorial mechanical thresholds
No BL differences were found (Fig. 5). In contrast, ANOVA revealed main effects of peri-sciatic drug (_F_2,120=35.364, P<0.0001), territory (_F_1,120=17.185, P<0.0001) and laterality (_F_4,120=2.406, P<0.05) as well as a reliable interaction between peri-sciatic drug and laterality (_F_2,120=2.406, P<0.05). Post hoc comparisons revealed that peri-sciatic vehicle had no effect on von Frey responses in either paw or territory (Fig. 5A). Four micrograms zymosan lowered mechanical response thresholds in the left (ipsilateral) sciatic and saphenous territories equally. These response thresholds were reliably lower than those recorded from the contralateral sciatic and saphenous territories (Fig. 5B) (P values <0.05). Unlike Experiment 2, 4 μg zymosan produced a mild allodynia in the contralateral hindpaw at 24 h (P<0.05). Again, the effects in the right saphenous and right sciatic nerves were statistically comparable. After 40, 160, and 400 μg zymosan (Fig. 6C,D,E), bilateral allodynias were observed in both the sciatic and saphenous territories, as both left and right sciatic and saphenous regions exhibited lowered mechanical thresholds compared to vehicle controls (P<0.0005). The observation of extra-territorial, in addition to territorial, allodynia in the ipsilateral and/or contralateral hindpaws suggests a central level of sensitization.
Territorial and extra-territorial allodynia produced by peri-sciatic zymosan. Top left drawing illustrates the saphenous (striped) and sciatic (white) regions of innervation of the rat hindpaw. Adapted from Tal and Bennett (1994). In each panel, data for the left sciatic nerve are represented by white squares, for the right hindpaws by black squares, for the left saphenous by white diamonds, and for the right saphenous by black diamonds. (Panel (A)) Vehicle injection around the left sciatic nerve had no effect on von Frey responses by any paw or innervation zone. (Panel (B)) 4 μg zymosan injection around the left sciatic nerve reliably reduced the response thresholds of both the sciatic and saphenous innervation zones in the left hindpaw, but did not reliably affect either innervation area of the right paw. (Panels (C, D, E)) 40, 160, and 400 μg zymosan injected around the left sciatic nerve reliably reduced the response thresholds of both the sciatic and saphenous innervation zones in both the left and right hindpaws.
Lack of effect of peri-sciatic zymosan on thermal response thresholds tested 24 h after injection. In each panel, data for left hindpaws are represented by white squares and for right hindpaws by black squares. Equivolume vehicle (Panel (A)), 4 μg zymosan (Panel (B)), 40 μg zymosan (Panel (C)), 160 μg zymosan (Panel (D)), and 400 μg zymosan (Panel (E)) did not reliably affect thermal thresholds of either hindpaw 24 h after peri-sciatic injection.
3.5. Experiment 5: assessment of the potential effects of peri-sciatic zymosan on thermal responsivity 24 h later
No BL differences were observed (Fig. 6). No dose of peri-sciatic zymosan reliably affected the thermal response latencies of either the left or right paws, compared to vehicle controls.
3.6. Experiment 6: assessment of the potential effects of peri-sciatic zymosan on thermal responsivity 1, 3, and 24 h later
Low threshold mechanical allodynia measurements at 3.5 and 24.5 h after peri-sciatic injection confirmed that 40 μg zymosan was effective in producing bilateral mechanical allodynia (Fig. 7B). ANOVA revealed the main effects of peri-sciatic drug (_F_1,14=42.811, P<0.0001) and time (_F_1,14=5.943, P<0.05), and an interaction of peri-sciatic drug and time (_F_1,14=4.843, P<0.05). Post hoc comparisons indicated that 40 μg zymosan induced bilateral allodynia relative to vehicle controls. That is, the left and right hindpaws of zymosan-injected rats did not differ in their thresholds but each hindpaw of the zymosan group exhibited lower thresholds than the hindpaws of the vehicle control group (P<0.01).
Lack of effect of peri-sciatic zymosan on thermal response thresholds tested 1, 3, and 24 h after injection. In each panel, data for left hindpaw after peri-sciatic vehicle are represented by white squares, for right hindpaw after peri-sciatic vehicle by black squares, for left hindpaw after peri-sciatic 40 μg zymosan by white diamonds, and for right hindpaw after peri-sciatic 40 μg zymosan by black diamonds. (Panel (A)) Zymosan did not reliably affect thermal responses at any time for either hindpaw, compared to vehicle controls. (Panel (B)) Verification that, in these same animals, low threshold mechanical allodynia was expressed. The animals whose data are shown in (A) were also tested for low threshold mechanical response thresholds at 3.5 and 24.5 h after injection, that is, shortly after thermal responsivity measures were completed on day 1 and on day 2. Bilateral allodynia was again produced by 40 μg zymosan compared to vehicle controls.
In contrast to the effectiveness of zymosan in altering mechanical thresholds, zymosan had little if any effect on thermal responsivity. No BL differences were observed. Also, ANOVA revealed that zymosan failed to reliably affect the thermal response latencies of either the left or right paws, compared to vehicle controls. These results support and extend the data from Experiment 5. Thus, no evidence was found that peri-sciatic zymosan alters thermal thresholds at a dose that produces robust bilateral allodynia.
4. Discussion
These studies demonstrate that zymosan (yeast cell walls) focally injected around a single sciatic nerve creates dose-dependent unilateral and bilateral low threshold mechanical allodynia, but no apparent thermal hyperalgesia. We have termed this phenomenon sciatic inflammatory neuritis (SIN). Low (4 μg) dose zymosan induces unilateral hindpaw allodynia, ipsilateral to the injection. Higher (40–400 μg) doses of zymosan induce bilateral hindpaw allodynia. Failure of zymosan (40 μg) to induce allodynia when injected into adjoining muscle suggests that immune activation must occur in close proximity to nerves for pain changes to occur. Allodynia is not specific to zymosan, as the proinflammatory cytokine HMG (Wang et al., 1999) also dose-dependently creates unilateral and bilateral hindpaw mechanical allodynia. Regarding the allodynias induced by zymosan, the bilateral allodynic effects do not appear to be explained by spread to the systemic circulation since: (a) equivolume dye remains confined to the gelfoam at least at the 0.5-h time tested, (b) injection of zymosan into adjoining muscle has equal potential for systemic spread yet fails to induce allodynia in any paw, (c) injection of even high doses of zymosan (400 μg) fails to induce allodynia at distant body regions (front paws), and (d) draining and distant immune tissues are not enlarged, suggesting that zymosan does not reach these sites. As such, the results agree with the proposal of Koltzenburg et al. (1999) that humoral mechanisms are unlikely to account for ‘mirror’ phenomena.
In addition, the present study demonstrates that peri-sciatic zymosan causes allodynia not only in skin innervated by the sciatic nerve (that is, territorial allodynia) but also in skin innervated by the saphenous nerve (that is, extra-territorial allodynia). Since no peripheral communication pathways are known to exist between these nerves (Tal and Bennett, 1994), the observation of extra-territorial saphenous allodynia strongly supports a central site of sensitization. Intriguingly, the ‘mirror’ allodynia observed in the right hindpaw after higher doses of zymosan over the left sciatic nerve also shows both ‘territorial’ (sciatic) and ‘extra-territorial’ (saphenous) mirror distributions. While various neural mechanisms have been considered to account for ‘mirror’ phenomena (for an excellent review, see Koltzenburg et al., 1999), the rapidity with which the ‘mirror’ effects occur in response to SIN suggests that a novel mechanism may underlie the present phenomena.
The most likely explanation for all of these pain changes is immune activation in close proximity to the sciatic nerve. These immune changes must occur rapidly in response to zymosan as robust unilateral and bilateral allodynias were observed within 3 h in the present study (the earliest time tested) and, in fact, within 1 h in more recent experiments where a more complete timecourse was examined (Watkins et al., 2001a,b). The speed of allodynia onset negates its mediation by previously proposed mechanisms (Seltzer et al., 1991; McLachlan et al., 1993; Yamamoto and Yaksh, 1993; Mannion et al., 1996; Neumann et al., 1996; Liu et al., 2000a).
It is clear that peripheral nerves can suffer ‘innocent bystander’ damage as a result of nearby immune activation that is not specifically directed at the nerves. Indeed, nerve damage results from their injection with activated macrophages (Said and Hontebeyrie-Joskowicz, 1992) or foreign proteins to which the host's immune cells have been sensitized (Wisniewski and Bloom, 1975; Harvey et al., 1995). There are numerous immune cell-derived candidate mediators that could account for the rapid appearance of SIN-induced allodynia, including proinflammatory cytokines, reactive oxygen species (ROS), and membrane attack complexes (MACs). Proinflammatory cytokines may be involved since injection of peripheral nerves with either TNF (Redford et al., 1995) or IL6 (Deretzi et al., 1999) damages the nerve and enhances pain (see Section 1). ROS are implicated in very early stages of inflammation-related nerve damage (Muijsers et al., 1997; Smith et al., 1999) and cause increased neuronal excitability (Mattson, 1998) and pain (Khalil et al., 1999). Lastly, MACs formed via complement cascade activation (Daha et al., 1979) may potentially be involved since MACs punch holes in axonal membranes, forming non-selective ion pores (Koski, 1992). MACs may mediate zymosan effects since zymosan is an activator of the alternative complement cascade that forms MACs (Daha et al., 1979).
The dose-dependent shift from unilateral (ipsilateral to the site of injection) to bilateral (both hindpaws) allodynia is intriguing since, as reviewed above, this shift does not appear to be accounted for by systemic spread of zymosan to the general circulation. Although preliminary, recent evidence from our laboratory suggests that the change from unilateral to bilateral (‘mirror’) allodynia is correlated with: (a) ROS and proinflammatory cytokine production around the sciatic nerve, (b) recruitment of immune cells to the area, and (c) morphological changes (circumferential edema of the outer regions of the nerve bundle) within the sciatic nerve (Watkins et al., 2001a,b). The central changes that account for the shift from SIN-induced unilateral to bilateral allodynia are unknown, although various neuronally based hypotheses have been suggested (Koltzenburg et al., 1999). Preliminary evidence from our laboratory suggests that glial activation and spinal release of the proinflammatory cytokine IL1 mediate the appearance of ‘mirror’ phenomena (Watkins et al., 2001a,b). From the literature, what is known is that bilateral pain changes have been previously observed with neuropathies. ‘Mirror’ pain has been reported clinically in causalgia and reflex sympathetic dystrophy (for review, see Seltzer and Shir, 1991). It has also been observed in various animal models of neuropathic pain (Seltzer et al., 1990; Kim and Chung, 1992; DeLeo et al., 1994; Tal and Bennett, 1994). As noted previously, these models involve local immune activation in addition to nerve trauma. Notably, nerve trauma is not required for mirror phenomena to occur since knee joint inflammation (Rees et al., 1996) and subcutaneous inflammation (Aloisi et al., 1993) have been reported to induce bilateral changes in pain.
SIN-induced mirror pain and extra-territorial pain provide evidence that central sensitization is occurring. Mirror pain within the same nerve (e.g. sciatic) territory of the contralateral limb is more frequently examined in the literature than is extra-territorial pain. Both intra-spinal and supra-spinal centrifugal pathways have been proposed to account for mirror effects (Watkins and Maier, 1997). Pain changes in the territories of nearby nerves in the ipsilateral limb are generally assumed to reflect intra-spinal rather than descending modulation (Tal and Bennett, 1994). While the saphenous and sciatic nerves enter the spinal cord separately (L3 and L4–L6 spinal roots, respectively) (Yoon et al., 1996) and maintain distinct somatotopic representations in the spinal cord dorsal horn, these somatotopic representations are in very close proximity throughout spinal segments L2–L5 (Swett and Woolf, 1985). Indeed, the subdivisions of the sciatic nerve each project to a distinct region of the dorsal horn and collectively form a ‘U’ shaped zone of terminal labeling, with the gap of the ‘U’ occupied by the terminals of the saphenous nerve (Swett and Woolf, 1985). This would allow the possibility that spinal mediators released in the dorsal horn sciatic nerve terminal region diffuse to the adjoining saphenous representation, affecting sensory processing there as well.
The one previously published report of peri-sciatic immune activation creating enhanced pain states is the work of Eliav et al. (1999). To create immune activation, these investigators applied killed bacteria (the active component of complete Freund's adjuvant) or carrageenan (seaweed protein) to the sciatic nerve at the time of surgery under pentobarbital anesthesia. Like low dose zymosan, killed bacteria and carrageenan induced a unilateral mechanical allodynia. Unlike zymosan, killed bacteria and carrageenan also induced a unilateral thermal hyperalgesia. These pain changes were generally observable 1 day later (no earlier time was tested), reaching a maximal effect at 2–3 days after surgery. At the doses of killed bacteria and carrageenan used, no ‘mirror’ effects were observed. While both Bennett's laboratory and our own are pursuing the changes that occur at the level of the nerve, it is unclear at present what may be found to account for the differences in behavioral outcomes observed.
In conclusion, acute immune activation in close proximity of the healthy sciatic nerve trunk induces territorial and extra-territorial mechanical allodynia in the apparent absence of thermal hyperalgesia. While lower levels of immune activation alter mechanical thresholds of only the injected leg, higher levels of immune action create ‘mirror’ allodynia as well. Such observations may have direct implications for clinical neuropathies resulting from inflammation and infection of otherwise healthy peripheral nerves. This is because the effects observed here for acute immune activation would be expected to persevere as immune activation is chronically maintained. The recognition that immune activation in close proximity to nerve bundles can have profound effects on somatosensory processing raises the possibility that immunomodulators can provide novel approaches to pain control.
Acknowledgements
This work was supported by NIMH grants MH45045, MH01558, and MH50479; NIH grant NS38020; the University of Colorado Undergraduate Research Opportunities Program; and the Hughes Initiative for Undergraduate Research.
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Keywords:
Neuropathy; Zymosan; Thermal hyperalgesia; Hargreaves test; von Frey test; High mobility group-1
© 2001 Lippincott Williams & Wilkins, Inc.