Leukemia inhibitory factor induces mechanical allodynia but not thermal hyperalgesia in the juvenile rat

Neuroscience Vol. 71, No. 4, pp. 1091-1094, 1996

Pergamon

Elsevier Science Ltd Copyright © 1996 IBRO Printed in Great Britain. All rights reserved 0306-4522/96 $15.00 + 0.00

0306-4522(95)00537-4

LEUKEMIA INHIBITORY FACTOR INDUCES M E C H A N I C A L A L L O D Y N I A BUT N O T T H E R M A L H Y P E R A L G E S I A IN T H E J U V E N I L E R A T S. W. N. THOMPSON*:~, A. D R A Y t a n d L. U R B A N Sandoz Institute for Medical Research, London and *Division of Physiology, UMDS, St Thomas' Campus, Lambeth Palace Road, London SE1 7EH, U.K. Abstract--Systemic administration or local injection to the rat hindpaw of leukemia inhibitory factor induced a prolonged, dose dependent, mechanical hypersensitivity of the hindpaw flexion withdrawal reflex. Mechanical stimuli which were innocuous prior to leukemia inhibitory factor administration, evoked a rapid hindpaw withdrawal reflex indicative of mechanical allodynia. Pre-administration of anti-leukemia inhibitory factor antibodies prevented this behavioural hypersensitivity. Hindpaw sensitivity to a noxious thermal stimulus was unaffected by leukemia inhibitory factor administration. Anti-leukemia inhibitory factor had no effect upon hindpaw withdrawal thresholds when injected alone nor influenced the mechanical hypersensitivity produced by a subcutaneous injection of nerve growth factor. Injection of the closely related cytokine ciliary neurotrophic factor did not affect mechanical or thermal reflexwithdrawal thresholds. Elevation of the neuroactive cytokine leukemia inhibitory factor following peripheral nerve injury may be a contributory factor to the pathogenesis of neuropathic pain. Key words: allodynia, hyperalgesia, cytokine, neuropathic pain, nerve injury.

Peripheral nerve a x o t o m y induces rapid changes in the excitability o f injured sensory afferents including s p o n t a n e o u s activity a n d novel sensitivity to mechanical, t h e r m a l a n d chemical stimuli. 4'22 In a d d i t i o n to these early alterations in afferent excitability however, delayed changes frequently accomp a n y n e u r o p a t h i c injury. Significant alterations in the n e u r o c h e m i c a l p h e n o t y p e o f p r i m a r y afferents 9 m a y underlie functional changes within the spinal cord, such as inability o f the cut afferent fibers to evoke p r i m a r y afferent depolarization, 21 somatotopically i n a p p r o p r i a t e responses 5'1° a n d the a p p e a r a n c e of novel receptive field properties, l° These functional a n d n e u r o c h e m i c a l changes m a y be m i m i c k e d by i n t e r r u p t i o n in a x o n a l t r a n s p o r t from the periphery 6 a n d w h e n afferent fibres are forced to i n n e r v a t e i n a p p r o p r i a t e targets, l°'13 O n the o t h e r h a n d , s u p p l e m e n t a t i o n o f trophic substances such as nerve g r o w t h factor ( N G F ) m a y specifically regulate the s t r e n g t h a n d location of synaptic inputs to the spinal cord. II These findings suggest t h a t trophic factor availability m a y influence the central connectivity o f sensory n e u r o n s a n d m a y be involved in the plasticity o f central synaptic transmission following peripheral injury.

Peripheral nerve injury results in alterations in the regulation o f several neuroactive cytokines a n d their receptors. 2'17 U n d e r n o r m a l circumstances, for instance, the levels o f leukemia inhibitory factor (LIF), a cytokine related to ciliary n e u r o t r o p h i c factor ( C N T F ) , are very low in the peripheral nervous system. Following sciatic nerve injury however, L I F expression is increased b o t h at the site o f injury a n d within the dorsal root ganglia ( D R G ) . 2 L I F is retrogradely t r a n s p o r t e d to the D R G following application to a peripheral nerve or target tissue, 3"7 is responsible for the i n d u c t i o n o f substance P, vasoactive intestinal polypeptide V I P (VIP) a n d galanin in superior cervical ganglion n e u r o n s following a x o t o m y 19 a n d will induce a n increase in VIP c o n t e n t o f D R G n e u r o n s in culture. 15 Transgenic studies have recently d e m o n s t r a t e d a n i m p o r t a n t function for this cytokine in the response to nerve i n j u r y ) 9 It is clear t h a t L I F expression is induced by nerve injury a n d is i m p o r t a n t for the n o r m a l response o f the nervous system to injury. T o investigate w h e t h e r L I F has a p r i m a r y role in the pathogenesis o f n e u r o p a t h i c injury we have studied the effects o f artificially raising the levels o f L I F u p o n the sensitivity o f the nociceptive h i n d p a w flexion w i t h d r a w a l reflect in y o u n g rats.

tPresent address: Astra Pain Research Unit, 275 Boul. Ammand-Frappier, Edifice 3000, Laval, Quebec, Canada, H7V 4A7 :~To whom correspondence should be addressed. Abbreviations: BSA, bovine serum albumin; CNTF, ciliary neurotrophic factor; DRG, dorsal root ganglia; LIF, leukemia inhibitory factor; NGF, nerve growth factor; PBS, phosphate-buffered saline; VIP, vasoactive intestinal polypeptide.

EXPERIMENTAL PROCEDURES Behavioural assessments were made of mechanical and thermal thresholds for the hindlimb nociceptive flexion withdrawal reflect in young rats (postnatal day 12-14). Mechanical thresholds were measured using calibrated yon Frey filaments ranging between 1.66 g and 14.75 g. Mechanical allodynia was quantified by measuring the percentage occurrence of hindlimb withdrawal in response to normally

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innocuous stimuli. Stimuli were applied with von Frey filaments ranging from 1.66 g to 12.6 g (1.66 g, 275 g, 4.57 g, 7.58 g, 12.6 g) to the dorsal surface of the left hindpaw of unrestrained rats. Each trial consisted of five applications of the von Frey filament within a 2 s period and each trial was repeated five times over a 5-10 min period. A set of five trails represented a single time point for each experimental group of animals. The occurrence of hindlimb withdrawal in each of these five trails could be expressed as a percentage response frequency (percent response frequency for each animal = number of foot withdrawals/5 × 100). Thermal thresholds were measured as the latency of hindlimb flexion from a radiant heat beam focused upon the plantar surface of the foot. Murine LIF; Anti-murine LIF neutralizing antibody and recombinant rat CNTF (all R & D Systems Abingdon, UK) were delivered in phosphate-buffered saline (PBS) containing 50/~g bovine serum albumin (BSA) per 1 #g cytokine, either in a 10/d subcutaneous injection to the left hindpaw (5 #1: dorsal surface, 5 #1: plantar surface) or adminis-

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Fig. 1. Behavioural assessments were m a d e of mechanical and thermal thresholds for the hindlimb nociceptive flexion withdrawal reflex in young rat pups (postnatal day 12-14). Mechanical thresholds were measured using calibrated von Frey filaments. Thermal threshold was measured as the latency of hindlimb flexion from a radiant noxious heat beam focused upon the plantar surface of the foot. All substances were administered systemically (100/~1, i.p.) in PBS containing 5 0 / t g BSA per 1 ,ug cytokine, or in a 10-ul subcutaneous injection to the left hindpaw (5 #1: dorsal Surface, 5 ,ul: plantar surface) or administered systematically (100/d, i.p.). Statistical evaluation was carried out using Student's t-test assuming equal variance. All values in the text are expressed as m e a n _+ S.E.M. Systemic injection of leukemia inhibitory factor (LIF) induces mechanical but not thermal behavioural hypersensitivity. (A) A single injection of L I F at time zero (1000 ng) induced a rapid and long-lasting hypersensitivity to mechanical stimuli. G r a p h shows the change in von Frey threshold required to evoke hindpaw flexion withdrawal following either L I F injection or vehicle (BSA in PBS). A drop in threshold was apparent 1.0 h following L I F injection which remained significant up to 4 h following injection of the cytokine. Vehicle was without effect. (B) The latency of hindpaw withdrawal from a noxious thermal stimulus in the same group of animals was not altered by systemic L I F injection. **P < 0.01, t-test, n = 8.

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Fig. 2. (A) Subcutaneous injection of L I F to the left hindpaw (time 0) produced a dose-dependent decrease in the mechanical threshold for hindpaw withdrawal. At the highest dose (1000 ng, filled squares) this decrease was significant 1 h following injection and remained significant for 24 h following injection. The effect of vehicle injection was transient and without significance (filled triangles). **P < 0.01, *P < 0.05, t-test, n = 8. (B) The mechanical hypersensitivity induced by L I F (1000 ng) was prevented by pre-administration of anti-LlF (0.66mg) (filled squares). Anti-LIF alone or the related cytokine C N F T had no significant effect upon hindpaw withdrawal thresholds (empty circles, diamonds, respectively). A single subcutaneous injection of nerve growth factor (1000 ng) induced delayed mechanical hypersensitivity which was not prevented by pretreatment with anti-LlF antibodies (empty triangles). **P < 0.01, t-test, n = 8.

tered systemically (100/~1 i.p.). NGF (2.5 s, Promega), was delivered in PBS. RESULTS

A single systemic injection of murine L I F (1000 ng, i.p.) induced a significant decrease in the von Frey threshold required to evoke flexion of the left hindlimb (Fig. 1A). A significant reduction in mechanical threshold was present (3.94 + 0.55 g vs 6.07 + 0.27 g before injection, P < 0.01, n = 8), 2 h (3.67 + 0.4 g, P < 0 . 0 1 , n = 8 ) and 4 h (3.38___0.43g, P < 0 . 0 1 , n = 8) following L I F injection. The sensitivity persisted for 8 h and was fully resolved 24 h following injection. The sensitivity to a noxious thermal stimulus was unaltered at any time point following L I F administration (12.1 _+ 1.12 s latency before injection vs 12.2 + 1.06 s, 4 h following LIF; Fig. 1B). There was no change in mechanical or thermal sensitivity after vehicle treatment (Fig. 1). Subcutaneous injections of 100 and 1000ng (in 10 ~1) L I F to the hindpaw produced a prolonged and dose-dependent decrease in the yon Frey threshold required to evoke ipsilateral hindlimb flexion

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Time following injection (Hours) Fig. 3. Responses to repeated mechanical stimuli with the two weakest von Frey filaments used (2.75g and 1.66g). Each point represents the mean ___S.E.M. (in percentage) of the response frequency for groups of four rats. Two groups received LIF (100 ng and 1000 ng), one vehicle (50/~g BSA in PBS) and one served as naive control. The presence of mechanical allodynia is indicated by the increase in per cent response frequency to the von Frey filaments of very low bending force. (A) In naive animals, the 2.75 g filament rarely evokes a flexion withdrawal response. Between 2 and 8 h following the highest dose of LIF injection however, animals responded to approximately 90% of trials with this filament. The effect of vehicle injection was transient and without significance. (B) Naive animals never responsed to a filament of 1.66 g. This represents a very weak innocuous mechanical stimulus. Following LIF injection however animals responded to approximately 60% of trials with this filament.

(Fig. 2A). A significant difference was present 2 h following LIF injection and was maintained at the same level for 7 h. The mean threshold for hindlimb flexion fell from 5.77 + 0.24 g in naive animals to 3.91 _ 0.17 g (I00 ng LIF) and 2.28 _+0.14 g (1000 ng LIF) 2 h following injection (P < 0.01 t-test) and to 2.24 _+ 0.27 g (1000 ng LIF) 7 h following injection. A significant difference was still present 24 h following the highest dose of LIF when compared to naive (4.56 _ 0.88 vs 6.52 ___0.29 g, 1000 ng LIF, P < 0.05 t-test). Vehicle injection (50/~g BSA in PBS) was without significant effect. The sensitizing effect of LIF to innocuous mechanical stimuli was prevented by pre-treatment with an equimolar concentration of monoclonal anti-LIF (Fig. 2B). Anti-LIF had no effect upon hindpaw withdrawal thresholds when injected alone nor influenced the mechanical hypersensitivity produced by a subcutaneous injection of N G F (1000 ng; 10 #1) (3.52___0.49 g 7 h following N G F vs 8.08 + 1.66 g naive; Fig. 2B). Subcutaneous injection of the closely related cytokine CNTF (1000 ng; 10 #1) did not significantly alter the mean von Frey threshold for hindpaw withdrawal at any time point following injection. (10.09 _ 1.45 g before

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injection vs 7.45 ___0.95 g; 2 h after CNTF injection Fig. 2B.) The mechanical threshold for hindlimb flexion withdrawal following subcutaneous LIF injection falls within the range of innocuous stimuli for naive animals and was indicative of mechanical allodynia. To quantify mechanical allodynia, the percentage occurrence of hindlimb withdrawal in response to normally innocuous stimuli was measured (See Experimental Procedures). Figure 3 shows the response of four groups of rats to stimulation with the two weakest yon Frey filaments (1.66 g and 2.75 g). In the naive group of animals stimulation with either of these filaments rarely evoked hindlimb flexion (one positive trial with 2.75 g filament from a possible 120 trials over a 24 h period). Following subcutaneous injection of 1000 ng the ipsilateral hindpaw became very sensitive to this stimulus. Between 2 and 8 h following injection animals responded to approximately 90% of trials with the 2.75g filament (Fig. 3A). Naive animals do not respond to a filament of 1.66 g. However, animals responded to approximately 60% of trials with this filament between 2 and 7 h following 1000 ng LIF injection (Fig. 3B). DISCUSSION

LIF is known to be a polyfunctional cytokine which shares many properties with CNTF in its actions upon neurons. Chronically administered at high doses (2 #g three times daily) LIF is toxic with effects upon haematopoietic tissue and a variety of other target sites. ~4This is the first study however to demonstrate the acute effect of a single dose of LIF upon behavioural nociceptive reflect thresholds. Moreover, a modality-specific effect was apparent with LIF affecting mechanical sensitivity and not thermal reflex thresholds. Although widespread effects on a variety of functional systems have been previously observed ~4 our results suggests a more specific mechanism of action. At present the mechanism of action of LIF is unclear, however several possibilities exist. LIF may selectively alter the peripheral sensitivity of sensory neurons, either directly though the activation of neuronal LIF receptors or indirectly via immune mediators. Sensory neurons express high affinity receptor sites for LIF 7 and retrogradely transport LIF to the DRG, 3'7 it is not clear at present, however, if receptors are restricted to specific subpopulations of neurons or whether they are widely distributed. Nor is there any evidence at present to indicate the involvement of LIF receptor binding in peripheral sensitization. The LIF receptor is also present on monocytes/macrophages.8 It is possible therefore that mediators released from these cells in response to increased LIF levels could sensitize peripheral receptors.18 In relation to the present findings, however, sensory neurone sensitization has been found by most investigators to be rather modality

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specific. While sensitization to heat or algesic chemicals is prominent after injury to skin, mechanical thresholds remain largely stabte.~2 The likelihood that present findings may be explained by such peripheral sensitization is therefore low. There is a body of evidence to show that LIF interacts with sympathetic neurons in vivo. Thus LIF can induce neurotransmitter switching of sympathetic neurons ~ and is responsible for axotomy-induced changes in neuropeptides. 19The possibility of a LIFmediated interaction between the sympathetic and sensory nervous system remains to be investigated. Alternatively, LIF is known to be retrogradely transported to the dorsal root ganglion and exerts effects on gene expression in these cells.~7 Neurotransmitters and/or neuromediators may in turn be transported to the central terminals of primary sensory neurons. The consequence increase in presynaptic neurotransmitter availability will considerably amplify afferent input onto dorsal horn neurons and is likely to significantly influence central afferent connectivity23 and hence behavioural hypersensitivity. Reasons for the modality-specific effect observed in the present study are again unclear. The most parsimonious explanation for our observations would

be a LIF-induced change in neurotransmitter/ neuromodulator content of large diameter myelinated sensory fibres likely to respond to low threshold mechanical stimuli. A recent study has demonstrated novel induction of preprotachykinin mRNA in medium and large-sized ( > 2 , 0 0 0 # m 2) cells in the D R G following sciatic nerve axotomy, j6 The factor responsible for preprotachykinin gene induction in a population of neurons which did not normally express neuropeptides is unknown at present.

CONCLUSION

It is apparent that LIF levels rise dramatically in both D R G and peripheral nerve following axotomy. 2 Taken together with evidence from the present study where LIF injection in normal animals produces a behavioural hypersensitivity, the possibility arises that LIF may contribute towards the mechanisms which generate the hyperalgesia of neuropathic injuries. Acknowledgement--This work was partially supported by a grant from the Medical Research Council to S.W.N.T.

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