Pretreatment with gangliosides reduces abnormal nociceptive responses associated with a rodent peripheral mononeuropathy

Pain, 48 (1992) 391-396 0 1992 Elsevier Science

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Pretreatment with gangliosides reduces abnormal nociceptive responses associated with a rodent peripheral mononeuropathy Ronald

L. Hayes a, Jianren Mao ‘, Donald D. Price ‘, Antonino German0 Dominic0 d’Avella b and Mario Fiori ’ and D.J. Mayer ’

‘,

aMedical College of Virginia, MCV Station, Richmond, VA 23298 (USA), ’ Department of Neurosurgery, Uniuersity of Messina, Messina (Italy), and ’ Fidia Pharmaceuticals, PadoLla (Italy) (Received

5 March

1991, revision

received

18 July 1991, accepted

19 July 1991)

A peripheral mononeuropathy was produced in adult male rats by placing loosely constrictive Summary ligatures around the common sciatic nerve. As reported by others, this procedure reliably results in postoperative behavior indicative of hyperalgesia, allodynia, and potentially, spontaneous pain. In these experiments, thermal hyperalgesia was assessed by measuring foot-withdrawal latencies to radiant heat aimed at the plantar surface of rat hind paws. Behaviors potentially indicative of spontaneous pain were assessed by rating spontaneous hind paw guarding positions. Rats with sciatic nerve ligation were divided into 5 groups (n = 6/group). Three groups received injections (i.p.) of either 10, 20 or 40 mg/kg of cerebral ganglioside mixture, GA. The 4th group was injected with 10 mg/kg of the purified ganglioside GM,, and the 5th group received an equal volume of saline. All injections were given daily for 2 days before surgery, the day of surgery and 9 days after surgery. All animals were behaviorally assessed for 2 days prior to surgery, the day of surgery, as well as 1,3,5, 7, and 10 days afterwards. All 5 groups had significantly reduced latencies to hind paw withdrawal on the side ipsilateral to sciatic nerve ligation. However, these hyperalgesic responses were significantly attenuated in rats receiving GA or GM, pretreatment. These data suggest that this animal model of peripheral neuropathic pain is sensitive to pharmacological manipulations useful for understanding mechanisms of neuropathic pain, including mechanisms related to excitotoxic processes. Such studies could lead to development of clinical approaches to treat this disorder. Key words: Pain; Peripheral

neuropathy;

Gangliosides; Hyperalgesia;

Introduction Traumatic injuries to nerves sometimes result in characteristic symptoms of disordered pain perception. These include spontaneous pain and often one or more kinds of stimulus-evoked pain including intense pain when the affected skin is touched, cooled or warmed. The intensity of this evoked pain may increase with repeated stimulation (Price et al. 1989). We have a very incomplete understanding of the neuropathogenesis of disorders of pain sensation occurring when peripheral somatic nerves are damaged.

Correspondence to: R.L. Hayes, Ph.D., University of Texas Health Science Center at Houston, 6431 Fannin St., Suite 7.148, Texas Medical Center, Houston, TX 77030, USA.

Nociception; (Rat)

Therefore, it is encouraging that an animal model of neuropathic pain has recently been developed in rats (Bennett and Xie 1990). In this model, ligation of the common sciatic nerve results in chronic constrictive injury (CCI) and postoperative behavior indicative of hyperalgesia, allodynia and spontaneous nociception. These features strongly resemble those observed in human neuropathic pain disorders, such as reflex sympathetic dystrophy (Bonica 1979; Biasi and Rustioni 1988; Price et al. 1989). Additional behavioral assessments have confirmed these original observations (Attal et al. 1990). As with human neuropathic pain, mechanisms mediating these behavioral indices of neuropathic pain are unknown. However, the similarities of signs and symptoms across human neuropathic pain patients and the rat CC1 model suggest that the latter

392

could be extremely useful in investigating pharmacological interventions that serve to ameliorate neuropathic pain. Gangliosides are glycosphingolipids that occur in nearly all cellular membranes and are particularly concentrated in nervous tissue. These agents have been shown to be effective in treating features of a variety of diabetic and toxic peripheral neuropathies (Triban et al. 1989; Gregorio et al. 1990). Certain toxic neuropathies are also associated with the occurrence of pain symptoms (Bradley et al. 1970). Mechanisms of ganglioside action in peripheral nerves include enhancement of mean sprouting length (Robb and Keynes 19841, increased number of regenerating axons (Sparrow and Grafstein 1982; Mengs et al. 1986), and increased conduction velocity of crushed rat sciatic nerve (Menga et al. 1984). Ganglioside treatment can also reduce disturbances in compound action potentials associated with toxic neuropathy of the sciatic nerve (Gregorio et al. 1990). In addition, gangliosides have been implicated in modulating long-term alterations in central nervous system function, perhaps associated with glutamate receptor activation (Ledeen 1989). Gangliosides may also attenuate glutamate-induced neuronal injury (Favaron et al. 1988; Mahadik et al. 1988; Skaper et al. 1989) as well as central nervous system injury produced by ischemia (Tanaka et al. 1986; Karpiak et al. 1987) or mechanical trauma (Stein and Sobel 1988), both of which may involve glutaminergic excitotoxicity. The purpose of the present study was to determine if treatment with gangliosides initiated prior to injury influenced behavioral manifestations of neuropathic pain in rodents. In addition, we wanted to determine if different cerebral gangliosides were differentially effective in this model. We report here that ganglioside treatment significantly reduces pain-related behaviors in this model. These results provide the first opportunities for careful preclinical assessments of pharmacological approaches to the management of neuropathic pain in humans.

Methods

Subjects Adult male Sprague-Dawley rats (Hilltop) weighing 300-350 g were used. Animals were individually housed in cages with water and food pellets available ad libitum. The animal room was illuminated from 7.00 to 19.00 h.

Surgery Animals were intraperitoneally above the sciatic according to the

anesthetized with 50 mg/kg sodium pentobarbital (i.p.1. A small incision was made at the site just nerve and the common sciatic nerve was ligated method of Bennett and Xie (1990). Briefly, the

common sciatic nerve was exposed at the level proximal to the sciatic trifurcation. A nerve segment approximately S-7 mm long was scparated from the connective tissue, and 4 ligatures (4-O chromic gut) were made around the nerve each with a I-1.S mm interval. The ligatures were carefully manipulated to the extent that the nerve was just barely constricted, so that the circulation through the superficial epineural vasculature was retarded but not arrested when viewing with a microscope. Sometimes a small, brief twitch could be seen during ligature. The incision was closed with 4-O silk suture. All animals were injected postoperatively with potassium penicillin (30000 IU/ratl intramuscularly to prevent infection. All surgical and experimental procedures were approved by our institutional Animal Care Committee.

Drug regimens and rationale Two ganglioside regimens were used: (I) GA, a highly purified bovine brain ganglioside mixture (21% GM,, 40% GD,,, 16% GD,,. 2% Go, and 2% G,,,,; and (2) 100% GM,. GA or GM, was first dissolved in 1:l chloroform/methanol mixture and dried with nitrogen gas. The pre-dissolved gangliosides were then resuspended with saline to the final 0.5% solution under gentle agitation. The pH value was adjusted to 7.35. Animals were divided into 5 groups (GA: 10, 20, 40 mg/kg; GM,: 10 mg/kg; saline) with 6 rats in each group. GA or GM, was injected i.p. The same volume of saline was given to the saline-treated rats. All the injections were given daily for 2 days before surgery, the day of surgery, and 9 days after surgery. Comparable doses of GA and GM, were assessed. The daily 10 mg/kg dose was selected since this dose regimen has been shown to he active in other studies of rat sciatic peripheral neuropathy (Triban et al. 1989). We initiated drug treatment prior to therapy since the pharmacokinetics of these compounds are incompletely understood. Moreover, pretreatment regimens are commonly used in studies of pharmacological mechanisms of injury to the central nervous system (Hayes et al. in press) or peripheral nerves (Giulio et al. 1989).

Behacioral assessments Thermal hyperalgesia. This behavioral test assessed responses of rat hind paws to noxious thermal stimulation. In this test, the rat was placed in an open-top plastic cylinder (18 cm diameter x 22 cm high) on a glass plate. Care was taken to ensure that the rat hind paw was in contact with the glass plate. The radiant heat source was from a bulb, which was focused on the plantar surface of the rat hind paw. The foot-withdrawal latency was determined as the time from start of radiant heat to the withdrawal of the rat hind paw from the glass surface. The light intensity of the bulb was adjusted so that a baseline of 12-13 set for foot-withdrawal latency was obtained. The cutoff time was preset to IS set to avoid tissue damage. Five trials were made for each hind paw ipsilateral and contralateral to sciatic ligation. The person who conducted the test was blind with respect to treatment group. The average value of the 5 trials was used for statistical analyses. Animals were behaviorally examined for 2 days prior to surgery, the day of surgery and 3, 5, 7, and 10 days after surgery. Obsen,ation of spontaneous nocicepticr behaviors. In order to analyze quantitatively changes in spontaneous nociceptive behaviors, rats receiving either 40 mg/kg of GA or saline (n = 9) were assessed using a modified grading method originally derived from the formalin test rating system (Dubuisson and Dennis 1977). Each rat was allowed to move freely without any intervention within a plexiglass cylinder (19 x 19x30 cm), and was habituated for 5 min before the observation. Within a 5 min observation period, the total duration of any of the following behaviors was recorded: (1) placement of only the medial edge or the heel of a ligated hind paw on the ground: (2) elevation of the hind paw; and (3) licking or scratching of the hind paw. The duration over which a CC1 rat presented one or more of these 3 behaviors within the 5 min observation period was counted

393 by a stopwatch. The average statistical analysis.

I

score of three 5 min trials was used for

--O--

SALINE

W

40 mg/kg GA

T

Statistical analyses Absolute latencies of ipsilateral and contralateral foot withdrawal were analyzed in addition to difference scores (contralateral latencyipsilateral latency) for paw latencies in each rat using a repeated measures analysis of variance (ANOVA). Then, the Waller-Duncan K ratio t test was used to determine the source of differences among groups. These same statistical tests were applied to analyze the data derived from the spontaneous nociceptive behavior ratings.

Results “7

Thermal hyperalgesia

For Day 2 prior to ligation and on the day of surgery, neither ganglioside (GM, nor GA) had an effect on baseline hind paw withdrawal latencies, nor were there differences between ipsilateral and contralateral hind paw latencies to radiant heat. Mean differences in withdrawal latencies between the 2 hind paws were very close to zero for all 5 groups for 2 days prior to ligation (Fig. 1). Moreover, both nerve ligation and ganglioside administration had no effect on contralateral hind paw withdrawal latencies (ANOVA P > 0.05).

SALINE

‘__._.~.._

-

71

10 mglkg GM1

-

10 mg/kg GA 20 mgikg GA 40 mg!kg GA

-I!

,

1

.

I,

I

I

.

I

.2 DAYS

7

-2

AFTER

LIGATION

LIGATIC+d I-w-----

Fig. 1. The effects of GA and GM, on rat foot flick latencies to noxious thermal heat. Difference scores for latencies to withdrawal of hind paws contralateral and ipsilateral to sciatic nerve ligation are plotted for 2 days prior to surgery C-21, the day of surgery CO), as well as 3, 5, 7 and 10 days after surgery. Note that by 3 days after surgery treatment both with GA and GM, had significantly reduced difference scores compared to saline-treated rats (SAL). The reductions on Days 3, 5 and 10 were significantly greater for 20 and 40 mg/kg of GA than for 10 mg/kg of GA or GM,. x = P < 0.05; xx = P < 0.01; xxx = P < 0.001 (ANOVA) overall differences between groups. * = P < 0.05(Wailer-Duncan) compared to salinetreated controls. Vertical bars are statistical errors in this figure and in Fig. 2. Some have been omitted for clarity.

% LIGATION

DAYS 2

AFTER 4

LIGATION c

I3

‘O

~+---------~

Fig. 2. The effects of 40 mg/kg of GA on behaviors indicative of spontaneous nociception. GA significantly reduced spontaneous nociceptive-related behaviors on Days 3, 5 and 7 after ligation. * P < 0.01; ** P < 0.001 (ANOVA) compared30 saline-treated controls.

In contrast, sciatic nerve ligtition produced significant reductions in ipsilateral hind paw withdrawal latencies as early as 3 days after injury in saline-treated rats as well as rats .treated with 10 mg/kg of GA or GM, which persisted for the 10 day observation period (P < 0.05; Wailer-Duncan: Day - 1 to Day 0 vs. Days 3-10). However, as shown in Fig. 1, even the 10 mg/kg doses of GA or GM, significantly reduced these hyperalgesic responses, as compared to saline-treated rats (P < 0.05, Waller-Duncan). Twenty and 40 mg/kg of GA completely abolished thermal hyperalgesia (P < 0.05, Waller-Duncan: Day -2 to Day 0 vs. Days 3-10). Groups receiving 10 mg/kg of GA or GM, did not significantly differ from each other. Moreover, 20 and 40 mg/kg of GA produced significantly greater reductions in thermal hyperalgesia on Days 3, 5 and 10 (P < 0.05, Waller-Duncan) than 10 mg/kg of either GA or GM,. Spontaneous nociceptive behaviors

Saline-treated rats displayed behaviors indicative of spontaneous pain for the 10 day post-ligation observation period. Forty mg/kg of GA significantly and potently reduced these behaviors on Days 3-7 (P < 0.05, ANOVA), as is shown in Fig. 2.

Discussion

We have shown that daily treatment with gangliosides initiated prior to injury can significantly blunt enhanced responses to noxious thermal stimuli following sciatic nerve ligation. A number of observations make it probable that the effects of gangliosides on

nociceptive behavior were associated with amelioration of disordered pain perception in these animals. Extensive analyses of this model in a variety of laboratories has led investigators to conclude that sciatic nerve ligation in rats are reliably associated with disturbances in pain sensation that, in fact, resemble those seen in humans (Attal et al. 1990; Bennett and Xie 1990). Importantly, these investigators have reported reliable association between changes in response to thermal stimuli, as employed in this study, and other measures of nociceptive response. Generalized changes in pain responsitivity were supported in this study by our quantitative assessments of other behaviors, such as the tendency of rats to guard the paw ipsilateral to ligation, which may be indicative of spontaneous pain. Moreover, initial analyses of regional changes in glucose metabolism in the dorsal horns of neuropathic rats indicates that a 10 mg/kg dose of GM, significantly reversed the increased metabolic activity that probably is associated with spontaneous pain (Hayes et al. 1991). However, it will be important in future studies to employ independent electrophysiological and functional mapping techniques to confirm alterations in encoding of nociceptive information. The mechanisms mediating ganglioside influences on behavioral responses to noxious thermal stimuli are not known. It is unlikely that these changes were associated with any direct analgesic effect, since gangliosides had no influence on baseline responses to thermal stimulation. Since the relative roles of peripheral nerve and central nervous system changes in this model are also unknown, gangliosides may have had peripheral or central effects, or both. As pointed out above, gangliosides can promote morphological (Sparrow and Grafstein 1982; Robb and Keynes 1984; Mengs et al. 1986) and electrophysiological (Mengs et al. 1984; Gregorio et al. 1990) indices of recovery from periphcral neuropathy. Importantly, in a model of toxic peripheral neuropathy, ganglioside treatment was especially effective in protecting the population of highspeed conducting sensory fibers (Gregorio et al. 1990). It is also conceivable that chronic pain syndromes are associated with enduring functional alterations in central nervous system circuits encoding nociception. These functional changes may represent pathological expressions of processes similar to those seen in memory formation which involve receptor-mediated activation of secondary cellular processes by excitatory amino acids such as glutamate (also see Dubner 1991). Activation of the N-methyl-D-aspartate (NMDA) receptor by glutamate contributes to induction of synaptic plasticity accompanying learning and memory (Collingridge and Bliss 1987). The resulting translocation of protein kinase C (PKC) from cytosol to membrane is a calciumdependent process which can mediate protein phosphorylation, possibly one of the most important post-

translational systems that regulates biological processes related to maintenance of changes in synaptic efficacy (Lynch et al. 1988; Linden and Routtenberg 1989). Excessive NMDA receptor activation by glutamate or other excitatory amino acids may also contribute to pathological, excitotoxic changes in neural function or cell death (Choi 1990; Hayes et al. 1990). The protection afforded by gangliosides in this study may be attributable to their ability to attenuate both in vivo (Mahadik et al.1988; Skaper et al. 1989) and in vitro (Favaron et al. 1988) neural injury induced by glutamate concentrations. Anatomical studies including immunohistochemical analyses indicate that glutamate (Westlund et al. 1989a; Westlund et al. 1990) (and possibly aspartate, see Stein and Sobel 1988) is a neurotransmitter in primary afferents including nociceptors terminating in superficial laminae of the rat dorsal horn (Biasi and Rustioni 1988). Glutamate is released by dorsal root stimulation in spinal cord slices (Jesse11 et al. 1986; Kawagoe et al. 1986). In vivo microdialysis studies ((Skilling et al. 1988) have also shown increased concentrations of glutamate and aspartate following intradermal injections of formalin. NMDA, quisqualate and AMPA (cr-amino-3-hydroxyS-methyl-4-isoxazola-propionic acid) primarily activate rat spinal nociceptive neurons (Aanonsen et al. 1990). Finally, intrathecal administration of NMDA can produce behavioral hyperalgesia (Aanonsen and Wilcox 1987) and intrathecal administration of a selective NMDA receptor antagonist (APS) can reduce behavioral responses to acute noxious stimuli (Cahusac et al. 1984). Since nerve transection produces massive discharges (Wall et al. 1974) and even deformation of axons can produce depolarization and neuronal firing (Julian and Goldman 19621, it is possible that chronic peripheral nerve constriction (e.g., sciatic nerve ligation) produces high-frequency discharges and glutamate release from terminals of nociceptive afferents. This release, in turn, may produce enduring changes in binding to receptor sites within the cation channel regulated by the NMDA receptor. Changes in PCP binding occur in dorsal horn laminae l/II of rats ipsilateral to ligation, persist for at least 10 days (Aanonsen et al. 1990) and may reflect a response of the NMDA receptor complex to pathologic levels of glutamate release (e.g., see Ledeen 1989). Finally, recent data from our laboratory show that blockade of the NMDA receptor can reduce behavioral indices of abnormal nociception in neuropathic rats (Hayes et al. 1991), and further suggest that activation of this receptor contributes to initiation and/or maintenance of neuropathic pain. The neuroprotective effects of gangliosides from excitotoxic injury may involve blockade of PKC translocation (Vaccarino et al. 1987). However, gangliosides can buffer intracellular calcium (Sillerud et al. 1979)

395

and modulate a variety of cellular events including the activity of several other protein kinases Waccarino et al. 1987). Thus, we have at present an incomplete understanding of changes in intracellular mechanisms potentially mediating the effect of gangliosides on neuropathic pain. Since sciatic nerve injury may also alter astrocytic function (Garrison et al. 1990), ganglioside influences may not be restricted to neurons. These initial studies detected no difference in the effects of equal doses of the ganglioside mixture, GA, and GM, alone. The biosynthesis and metabolism of gangliosides have been well described (Ledeen 1989). These initial observations suggest that GM, and/or its intermediate metabolites rather than other series of gangliosides may be sufficient to mediate the observed effects on nociceptive responses. Previous investigators have reported that morphine transiently reduces behaviors indicative of spontaneous pain (Mengs et al. 1986). However, the present study provides an indication of a possible pharmacological treatment the mechanisms of action of which are likely to differ from those of conventional analgesics (i.e., opiates). For example, no effects were observed on baseline nociceptive measures. It will be important to determine the longest time after injury at which initiation of ganglioside treatment will be effective. In addition to having important clinical implications, these experiments will provide important insight into the time course of mechanisms mediating the onset and maintenance of neuropathic pain. Further studies will be required to determine whether the effects of ganglioside are preferentially mediated by actions at peripheral or central loci. Finally, physiological and anatomical studies will be important to determine the changes in peripheral and central encoding of nociception associated with ganglioside treatment.

Acknowledgements The authors wish to acknowledge the assistance of Fay Akers in preparation of this manu~ript. This research was supported by a grant from Fidia and NS21458.

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