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Inhibitory effects of tachykinin receptor antagonists on thermally induced inflammatory reactions in a rat model
Burns 25 (1999) 125±129
Inhibitory eects of tachykinin receptor antagonists on thermally induced in¯ammatory reactions in a rat model O. LoÈfgren a, b, *, Y. Qi a, T. Lundeberg a, b a
Department of Physiology and Pharmacology, Karolinska Institute, 171 77 Stockholm, Sweden Department of Reconstructive Plastic Surgery, Division of Surgery and Rehabilitation, Karolinska Hospital, 171 76 Stockholm, Sweden
b
Accepted 29 July 1997
Abstract Recent studies have proposed that activation of the sensory nervous system after thermal injury induces the release of vasoactive neuropeptides, including tachykinins which contribute to the local in¯ammatory reaction as well as to the nociceptive transmission at the spinal cord level. Eects of the tachykinins substance P and neurokinin A are mediated by the neurokinin 1 and 2 (NK1, NK2) receptors. The aim of the present study was to investigate the modulatory role of NK1 and NK2 antagonists on edema formation, and on hindpaw withdrawal latency to experimentally asses nociception. Thermal injury was in¯icted on the anaesthetized rat by dipping the right hindpaw into hot water at 608C for 20 s. The amount of edema formation was calculated by measuring the hindpaw volume with a plethysmograph before and during 420 min after scalding. In other studies scalding was in¯icted under brief anesthesia, and hindpaw withdrawal latencies (HWL) to mechanical stimulation were recorded before injury and at 180 min after. The eect on edemic reactions of rats treated locally with NK1 and NK2 receptor antagonist were studied, as well as the eect of the same compounds on HWL after intrathecal injection. Scalding induced a progressive edema formation which was reduced signi®cantly in rats treated with local injection of 100 nmol of NK1 and NK2 antagonists 45 min after the injury. The thermally induced in¯ammation was followed by signi®cant decrease of the latency of hindpaw withdrawal response to mechanical stimulation. Intrathecal injection of 30 nmol of the same drugs 180 min after scalding was followed by signi®cant increase in HWL. The results indicate that SP and NKA contribute to the in¯ammatory reactions after thermal injury and that the tachykinin receptor antagonists possess the ability to reduce both the local edemic reaction as well as the nociceptive transmission at the spinal cord level. # 1999 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: Edema; Nociception; Sensory peptide antagonists
1. Introduction Thermal injury such as scalding commonly results in the activation of nociceptive aerents. Following their activation, a release of neuropeptides from the peripheral nerve endings occurs, contributing to the in¯ammatory reactions. Among the neuropeptides released are the tachykinins including substance P (SP) and neurokinin A (NKA) [1, 2]. The release of SP and NKA induces microvascular reactions such as vasodilatation and plasma extravasation which contribute to the edema formation [3, 4]. Furthermore, both neuropeptides are suggested to play a role in the trans-
* Corresponding author. Fax: +46-8-517-72505; e-mail: uke@ kir.ks.se
mission of nociceptive input at the spinal cord level [5]. The eects of SP and NKA are mediated by the neurokinin 1 and 2 (NK1, NK2) receptors [6]. Both receptors are widely distributed in the spinal cord as well as in peripheral tissue. The NK1 receptor is activated mainly by SP and the NK2 receptor by NKA. The expression of NK receptors has been shown to increase during an in¯ammatory process [7]. The role of tachykinins may be investigated through the use of selective peptide antagonists [8]. Recently, tachykinin receptor antagonists of nonpeptide origin have been synthesized and pharmacological potency is indicated [6]. The aim of the present study was to examine the contribution of tachykinins to thermally induced edema formation and the transmission of presumed painful sensation at the spinal cord level by using NK1 and NK2 antagonists.
0305-4179/99/$19.00 + 0.00 # 1999 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 9 8 ) 0 0 1 2 5 - 9
126
O. LoÈfgren et al. / Burns 25 (1999) 125±129
2. Material and methods All experiments were performed on freely moving male Sprague±Dawley rats (300±350 g; ALAB, Stockholm). The rats were housed in cages with free access to food and water, and maintained in a room temperature of 242 18 with a 12 h light/dark cycle. On the day of experimentation the rats were anaesthetized (see below) and after completion of testing immediately killed by an overdose. All experiments were approved by the local ethical committee at Karolinska Institute. 2.1. Model of thermally induced in¯ammation and edema measurement Eighteen rats were divided into three groups, according to drug treatment and a control group of 7 was given saline. The rats were ®rst anesthetized with chloralhydrate (0.4 g/kg) i.p. The thermal injury was achieved by dipping one hindpaw into water at 608C for 20 s while the other paw served as control. Hindpaw volumes were measured with plethysmometer (Ugo Basil 7150) before scalding and then continuously at intervals for 7 h afterwards. Test substances were administered by subplantar injection 45 min after thermal injury. 2.2. Test of the latency to withdrawal response Thermal injury was in¯icted under brief ether anaesthesia. Forty rats were divided in four groups, according to treatment with dierent NK1 and NK2 antagonists and another 40 treated with saline also divided: i.e. for each group of rats treated with an active compound there was one of similar size given saline. The Randall Selitto test (Ugo Basil 7200) was used to assess withdrawal latency to mechanical stimulation in awake animals. A wedge-shaped pusher with a loading rate of 48 g/s was applied to the dorsal surface of the manually handled hindpaw and the pressure required to initiate the struggle response was assessed. The hindpaw withdrawal latency (HWL) is expressed in seconds, i.e. latency to withdrawal from start of the stimulation. The HWL tests were performed before scalding, at 180 min after scalding immediately before the injection of the drug, and ®nally at 60 min after the injection. The lumbar skin was prepared with xylocain gel and intrathecal injection was performed through a stainless steel needle with an outer diameter of 0.5 mm inserted into the subarachnoid space between L4±L5 or L3±L4. Ten ml of solution (see Section 2.3) were thereafter infused during 1 min. All rats were accustomed to the testing conditions for at least 5 days before the initiation of the experiment in
order to decrease the stress induced by the measurements and to obtain a stable response latency. 2.3. Chemicals Solutions for subplantar injections were prepared with sterilized saline (0.9%), each with a volume of 0.1 ml: (i) 100 nmol of NK1 antagonist (Sendide, SC 916, Neosystem, France); (ii) 100 nmol of NK1 nonpeptidergic antagonist (L-703,606, RBI, USA); (iii) 100 nmol of NK2 antagonist (Men 10376, SC 438, Neosystem). Solutions for intrathecal administration were prepared with sterilized saline (0.9%), each with a volume of 10 ml: (i) 30 nmol of NK1 antagonist, (ii) 30 nmol of NK1 nonpeptidergic antagonist, (iii) 30 nmol of NK2 antagonist. Control groups received the same volume of saline. 3. Statistical analysis The paw volume measured in milliliters is analyzed by ANOVA with repeated measures design. The analysis is carried out for the four independent groups according to drug treatment. Time and thermal exposure conditions are dependent factors and tests for interactional eects were done to ®nd out if the changes over time were equal for all four groups and for scalded conditions versus control. Hindpaw withdrawal latency (HWL) after thermal injury was measured in seconds and paired t-test was used to determine dierences in HWL between scalded/control. A two factor ANOVA was used to estimate the change of HWL according to drug treatment and thermal exposure conditions. A p value below the a level of 0.05 is de®ned as statistical signi®cance. 4. Results 4.1. Eects of local injection of NK1 and NK2 antagonists on thermally induced edema formation In all groups the preburn volume was the same and there was no dierence between right and left hindpaws (Fig. 1). No edema formation was seen in the contralateral nonscalded paw during the time of recording. After scalding signi®cant ipsilateral edema formation was noted in all groups during the observation period ( p < 0.001). However, the plethysmogra®c volume measurements showed varying progressions of edema according to the treatment. The most prominent edema formation was observed in the group of rats which received saline The mean increase of paw volume during 0±420 min was 1.9 2 0.13 ml. Compared to the saline injected group, treatment with
O. LoÈfgren et al. / Burns 25 (1999) 125±129
127
Fig. 1. Paw volume (ml) change over time, 0±420 min after scalding in four dierent groups treated with local injection of NK antagonists and saline. No. 25.
the NK1 antagonist resulted in signi®cant reduction of edema formation over time ( p = 0.001). Injection of the NK2 antagonist also produced a reduction of edemic formation, but less pronounced ( p = 0.20). However, administration of the nonpeptidergic NK1 antagonist had no eect on the edema formation ( p = 0.51). The edema reaction was unrelated to initial paw volume. Such an eect has been allowed for by analyzing the correlation between initial value and change over time.
4.2. Eects of scalding on hindpaw withdrawal latency before and after intrathecal administration of NK1 and NK2 antagonists 180 min after scalding there was a signi®cant decrease in HWLs of scalded paws ( p = 0.004). However, the HWLs were signi®cantly decreased bilaterally (Fig. 2). Then 60 min after intrathecal injections HWLs increased among rats which received the NK receptor antagonists (Fig. 3). Compared to the saline
Fig. 2. Changes in hindpaw withdrawal latecy-HWL (s) to pressure after scalding of the right paw, 0±180 min. Figure illustrates four dierent groups before intrathecal injection with NK antagonists or saline. No. 80.
128
O. LoÈfgren et al. / Burns 25 (1999) 125±129
Fig. 3. Changes in hindpaw withdrawal latency (HWLs) to pressure after intrathecal injection of NK antagonists and saline in scalded rats. No. 80.
treated control group, the increase was signi®cant in the group of rats treated with the NK1 antagonist ( p = 0.039). Treatment with the NK2 antagonist was followed by a less pronounced increase of HWL ( p = 0.58). But after the injection of NK1 nonpeptidergic antagonist the increase of HWL was signi®cant ( p = 0.007). The eect of NK1 peptidergic and nonpeptidergic antagonist on HWLs was similar ( p = 0.97). In all groups treated with tachykinin antagonists, there was a tendency towards increases in HWLs on the contralateral side. (Fig. 3).
5. Discussion The results of the present studies show that the administration of antagonists directed towards receptors of the sensory neuropeptides SP (NK1 receptor antagonist) and NKA (NK2 receptor antagonist) in¯uence thermally induced in¯ammation. Unilateral scalding of the hindpaw was followed by a progressive ipsilateral edema formation which was signi®cantly reduced by local treatment with the NK1 antagonist. Administration of the NK2 antagonist also reduced the edema formation, although to a lesser extent. After scalding there was a bilateral decrease in hindpaw withdrawal latency to mechanical stimulation with the decrease more pronounced on the thermally injured paw. Intrathecal administration of the NK1 antagonist as well as the nonpeptidergic NK1 antagonist signi®cantly increased the withdrawal responses. The localized edema reaction after thermal injury is initiated by an early fall in interstitial hydrostatic pressure followed by transcapillary ¯uid shift [9] and the release of dierent in¯ammatory mediators [10]. In previous studies it has been reported that the sensory nervous system contributes to in¯ammatory processes, and collaborators have reported that SP is released
locally after scalding [11]. We have recently reported that another tachykinin, NKA, is released into the subcutaneous space of the paw after scalding [2]. Pretreatment with the neurotoxin capsaicin or with a substance P antagonist decreases plasma extravasation after scalding [12, 13]. In other studies, using an online laser Doppler technique, we reported a biphasic perfusion increase during the initial phase of the edema formation [14] which can be signi®cantly reduced by pretreatment with NK1 and NK2 antagonists [15]. This is in accordance with the present ®nding where the NK1 antagonist signi®cantly reduced the edema formation after thermal injury and the NK2 antagonist had a similar but less pronounced eect. Thermal injury results in the activation of primary nociceptive aerents followed by the release of SP and NKA at the dorsal horn of the spinal cord [4]. Duggan et al. suggested that NKA is especially involved in the transmission of noxious thermal stimuli [16]. In present studies intrathecally administered NK1 or NK2 peptidergic receptor antagonists resulted in an increase of hindpaw withdrawal responses, an eect most apparent after treatment with NK1 antagonists. Since there are drawbacks in the use of receptor antagonists of peptidergic origin, dierent nonpeptidergic antagonists have been developed during the last decade [6]. Therefore it was decided also to investigate the eect of a nonpeptidergic NK1 receptor antagonist in the present study. The result was a signi®cant increase in hindpaw withdrawal latency, equal in eect to the peptidergic one. As described above, unilateral scalding to the hindpaw resulted in an ipsilateral edema formation and a bilateral decrease in hindpaw withdrawal latencies to mechanical pressure. This is in line with other results, indicating a communication of sensory activity from one side to the other [17, 18]. As earlier mentioned it has been proposed that primary sensory neurons
O. LoÈfgren et al. / Burns 25 (1999) 125±129
contributes both to the local in¯ammatory reaction and the transmission of nociceptive information centrally [19]. The neurogenic contribution to the peripheral in¯ammatory reaction has been attributed to local axon re¯exes and dorsal root re¯exes, resulting in the release of sensory neuropeptides [20]. Recently it has also been shown that excessive nociceptive activation at the central terminals of primary aerents results in an interneuronal circuitry which may be followed by increased activity in contralateral nociceptive terminals [20]. Taken together, the results show that unilateral thermal injury causes an unilateral edema formation which is a part of a local defense reaction, whereas the bilateral increase of withdrawal latency is a sign of a general hyperalgesic response. Acknowledgements The study was supported by funds from the AnnaGreta Crafoord's Foundation, the Gustav Vth 80-year Anniversary Foundation, the Karolinska Institutet Foundation, the Magnus Bergwall Foundation, the Nanna Swartz Foundation and the Swedish Society against Rheumatism (RMR). References [1] Yonehara N, Shibutani T, Inoki R. Contribution of substance P to heat-induced edema in rat paw. J Pharmacol Exp Ther 1987;242:1071±6. [2] LoÈfgren O, Palmer B, Theodorsson E, ToÈrkvist L, Lundeberg T. Contribution of the sensory and sympathetic nervous system to scalding-induced edema in the rat paw. Burns 1998;24:188±95. [3] Siney L, Brain SD. Involvement of sensory neuropeptides in the development of plasma extravasation in rat dorsal skin following thermal injury. Br J Pharmacol 1996;117:1065±70. [4] Levine JD, Fields HL, Basbaum AI. Peptides and the primary aerent nociceptor. J Neurosci 1993;13:2273±86. [5] Wiesenfeld-Hallin Z, Xu XJ. The dierential roles of substance P and neurokinin A in spinal cord hyperexcitability and neurogenic in¯ammation. Regul Peptides 1993;46:165±73.
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[6] Quartara L, Maggi CA. The tachykinin NK1 receptor. Part II: distribution and pathophysiological roles. Neuropeptides 1998;32:1±49. [7] HoÈkfelt T. Neuropeptides in perspective: the last ten years. Neuron 1991;7:867±79. [8] Xu XJ, Dalsgaard CJ, Maggi CA, Wiesenfeld-Hallin Z. NK-1, but not NK-2, tachykinin receptors mediate plasma extravasation induced by antidromic C-®ber stimulation in rat hindpaw: demonstrated with the NK-1 antagonist CP-96,345 and the NK2 antagonist Men 10207. Neurosci Lett 1992;139:249±52. [9] Lund T, Onarheim H, Reed RK. Pathogenesis of edema formation in burn injuries. World J Surg 1992;16:2±9. [10] Arturson G. Pathophysiology of the burn wound and pharmacological treatment. The Rudi Hermans lecture, 1995. Burns 1996;22:255±74. [11] Haegerstrand A. Sensory neuropeptides in in¯ammation and cell proliferation. Ph.D. thesis, University of Stockholm, 1990. [12] Saria A, Lundberg JM. Capsaicin pretreatment inhibits heatinduced oedema in the rat skin. Naunyn-Schmiedeberg's Arch Pharmacol 1983;323:341±2. [13] Lundberg JM, Saria A, Rosell S, Folkers K. A substance P antagonist inhibits heat-induced oedema in the rat skin. Acta Physiol Scand 1984;120:145±6. [14] LoÈfgren O, Gazelius B, Lundeberg T. Acute microcirculatory changes after scalding of the rat paw. Acta Physiol Scand 1997;161:289±94. [15] LoÈfgren O, Qi Y, Lundeberg T, Gazelius B. Antagonists of sensory neuropeptides inhibit the secondary phase of increased circulation following thermally induced in¯ammation, Microv Res (In press). [16] Duggan AW, Hope PJ, Jarrott B, Schaible HG, FleetwoodWalker SM. Release, spread and persistence of immunoreactive neurokinin A in the dorsal horn of the cat following noxious cutaneous stimulation: studies with antibody microprobes. J Neurosci 1990;35:195±202. [17] Bileviciute I, Lundeberg T, Ekblom A, Theodorsson E. Bilateral changes of substance P-, neurokinin A-, calcitonin gene-related peptide- and neuropeptide Y-like immunoreactivity in rat knee joint synovial ¯uid during acute monoarthritis. Neurosci Lett 1993;153:37±40. [18] LoÈfgren O, Yu LC, Theodorsson E, Hansson P, Lundeberg T. Intrathecal CGRP8-37 results in a bilateral increase in hindpaw withdrawal latency in rats with a unilateral thermal injury. Neuropeptides 1997;6:601±7. [19] Mayer EA, Raybould H, Koelbel C. Neuropeptides, in¯ammation and motility. Dig Dis Sci 1988;33:71S±77S. [20] Sluka KA, Willis WD, Westlund KN. The role of dorsal root re¯exes in neurogenic in¯ammation. Pain Forum 1995;4:141±9.
Inhibitory eects of tachykinin receptor antagonists on thermally induced in¯ammatory reactions in a rat model O. LoÈfgren a, b, *, Y. Qi a, T. Lundeberg a, b a
Department of Physiology and Pharmacology, Karolinska Institute, 171 77 Stockholm, Sweden Department of Reconstructive Plastic Surgery, Division of Surgery and Rehabilitation, Karolinska Hospital, 171 76 Stockholm, Sweden
b
Accepted 29 July 1997
Abstract Recent studies have proposed that activation of the sensory nervous system after thermal injury induces the release of vasoactive neuropeptides, including tachykinins which contribute to the local in¯ammatory reaction as well as to the nociceptive transmission at the spinal cord level. Eects of the tachykinins substance P and neurokinin A are mediated by the neurokinin 1 and 2 (NK1, NK2) receptors. The aim of the present study was to investigate the modulatory role of NK1 and NK2 antagonists on edema formation, and on hindpaw withdrawal latency to experimentally asses nociception. Thermal injury was in¯icted on the anaesthetized rat by dipping the right hindpaw into hot water at 608C for 20 s. The amount of edema formation was calculated by measuring the hindpaw volume with a plethysmograph before and during 420 min after scalding. In other studies scalding was in¯icted under brief anesthesia, and hindpaw withdrawal latencies (HWL) to mechanical stimulation were recorded before injury and at 180 min after. The eect on edemic reactions of rats treated locally with NK1 and NK2 receptor antagonist were studied, as well as the eect of the same compounds on HWL after intrathecal injection. Scalding induced a progressive edema formation which was reduced signi®cantly in rats treated with local injection of 100 nmol of NK1 and NK2 antagonists 45 min after the injury. The thermally induced in¯ammation was followed by signi®cant decrease of the latency of hindpaw withdrawal response to mechanical stimulation. Intrathecal injection of 30 nmol of the same drugs 180 min after scalding was followed by signi®cant increase in HWL. The results indicate that SP and NKA contribute to the in¯ammatory reactions after thermal injury and that the tachykinin receptor antagonists possess the ability to reduce both the local edemic reaction as well as the nociceptive transmission at the spinal cord level. # 1999 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: Edema; Nociception; Sensory peptide antagonists
1. Introduction Thermal injury such as scalding commonly results in the activation of nociceptive aerents. Following their activation, a release of neuropeptides from the peripheral nerve endings occurs, contributing to the in¯ammatory reactions. Among the neuropeptides released are the tachykinins including substance P (SP) and neurokinin A (NKA) [1, 2]. The release of SP and NKA induces microvascular reactions such as vasodilatation and plasma extravasation which contribute to the edema formation [3, 4]. Furthermore, both neuropeptides are suggested to play a role in the trans-
* Corresponding author. Fax: +46-8-517-72505; e-mail: uke@ kir.ks.se
mission of nociceptive input at the spinal cord level [5]. The eects of SP and NKA are mediated by the neurokinin 1 and 2 (NK1, NK2) receptors [6]. Both receptors are widely distributed in the spinal cord as well as in peripheral tissue. The NK1 receptor is activated mainly by SP and the NK2 receptor by NKA. The expression of NK receptors has been shown to increase during an in¯ammatory process [7]. The role of tachykinins may be investigated through the use of selective peptide antagonists [8]. Recently, tachykinin receptor antagonists of nonpeptide origin have been synthesized and pharmacological potency is indicated [6]. The aim of the present study was to examine the contribution of tachykinins to thermally induced edema formation and the transmission of presumed painful sensation at the spinal cord level by using NK1 and NK2 antagonists.
0305-4179/99/$19.00 + 0.00 # 1999 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 9 8 ) 0 0 1 2 5 - 9
126
O. LoÈfgren et al. / Burns 25 (1999) 125±129
2. Material and methods All experiments were performed on freely moving male Sprague±Dawley rats (300±350 g; ALAB, Stockholm). The rats were housed in cages with free access to food and water, and maintained in a room temperature of 242 18 with a 12 h light/dark cycle. On the day of experimentation the rats were anaesthetized (see below) and after completion of testing immediately killed by an overdose. All experiments were approved by the local ethical committee at Karolinska Institute. 2.1. Model of thermally induced in¯ammation and edema measurement Eighteen rats were divided into three groups, according to drug treatment and a control group of 7 was given saline. The rats were ®rst anesthetized with chloralhydrate (0.4 g/kg) i.p. The thermal injury was achieved by dipping one hindpaw into water at 608C for 20 s while the other paw served as control. Hindpaw volumes were measured with plethysmometer (Ugo Basil 7150) before scalding and then continuously at intervals for 7 h afterwards. Test substances were administered by subplantar injection 45 min after thermal injury. 2.2. Test of the latency to withdrawal response Thermal injury was in¯icted under brief ether anaesthesia. Forty rats were divided in four groups, according to treatment with dierent NK1 and NK2 antagonists and another 40 treated with saline also divided: i.e. for each group of rats treated with an active compound there was one of similar size given saline. The Randall Selitto test (Ugo Basil 7200) was used to assess withdrawal latency to mechanical stimulation in awake animals. A wedge-shaped pusher with a loading rate of 48 g/s was applied to the dorsal surface of the manually handled hindpaw and the pressure required to initiate the struggle response was assessed. The hindpaw withdrawal latency (HWL) is expressed in seconds, i.e. latency to withdrawal from start of the stimulation. The HWL tests were performed before scalding, at 180 min after scalding immediately before the injection of the drug, and ®nally at 60 min after the injection. The lumbar skin was prepared with xylocain gel and intrathecal injection was performed through a stainless steel needle with an outer diameter of 0.5 mm inserted into the subarachnoid space between L4±L5 or L3±L4. Ten ml of solution (see Section 2.3) were thereafter infused during 1 min. All rats were accustomed to the testing conditions for at least 5 days before the initiation of the experiment in
order to decrease the stress induced by the measurements and to obtain a stable response latency. 2.3. Chemicals Solutions for subplantar injections were prepared with sterilized saline (0.9%), each with a volume of 0.1 ml: (i) 100 nmol of NK1 antagonist (Sendide, SC 916, Neosystem, France); (ii) 100 nmol of NK1 nonpeptidergic antagonist (L-703,606, RBI, USA); (iii) 100 nmol of NK2 antagonist (Men 10376, SC 438, Neosystem). Solutions for intrathecal administration were prepared with sterilized saline (0.9%), each with a volume of 10 ml: (i) 30 nmol of NK1 antagonist, (ii) 30 nmol of NK1 nonpeptidergic antagonist, (iii) 30 nmol of NK2 antagonist. Control groups received the same volume of saline. 3. Statistical analysis The paw volume measured in milliliters is analyzed by ANOVA with repeated measures design. The analysis is carried out for the four independent groups according to drug treatment. Time and thermal exposure conditions are dependent factors and tests for interactional eects were done to ®nd out if the changes over time were equal for all four groups and for scalded conditions versus control. Hindpaw withdrawal latency (HWL) after thermal injury was measured in seconds and paired t-test was used to determine dierences in HWL between scalded/control. A two factor ANOVA was used to estimate the change of HWL according to drug treatment and thermal exposure conditions. A p value below the a level of 0.05 is de®ned as statistical signi®cance. 4. Results 4.1. Eects of local injection of NK1 and NK2 antagonists on thermally induced edema formation In all groups the preburn volume was the same and there was no dierence between right and left hindpaws (Fig. 1). No edema formation was seen in the contralateral nonscalded paw during the time of recording. After scalding signi®cant ipsilateral edema formation was noted in all groups during the observation period ( p < 0.001). However, the plethysmogra®c volume measurements showed varying progressions of edema according to the treatment. The most prominent edema formation was observed in the group of rats which received saline The mean increase of paw volume during 0±420 min was 1.9 2 0.13 ml. Compared to the saline injected group, treatment with
O. LoÈfgren et al. / Burns 25 (1999) 125±129
127
Fig. 1. Paw volume (ml) change over time, 0±420 min after scalding in four dierent groups treated with local injection of NK antagonists and saline. No. 25.
the NK1 antagonist resulted in signi®cant reduction of edema formation over time ( p = 0.001). Injection of the NK2 antagonist also produced a reduction of edemic formation, but less pronounced ( p = 0.20). However, administration of the nonpeptidergic NK1 antagonist had no eect on the edema formation ( p = 0.51). The edema reaction was unrelated to initial paw volume. Such an eect has been allowed for by analyzing the correlation between initial value and change over time.
4.2. Eects of scalding on hindpaw withdrawal latency before and after intrathecal administration of NK1 and NK2 antagonists 180 min after scalding there was a signi®cant decrease in HWLs of scalded paws ( p = 0.004). However, the HWLs were signi®cantly decreased bilaterally (Fig. 2). Then 60 min after intrathecal injections HWLs increased among rats which received the NK receptor antagonists (Fig. 3). Compared to the saline
Fig. 2. Changes in hindpaw withdrawal latecy-HWL (s) to pressure after scalding of the right paw, 0±180 min. Figure illustrates four dierent groups before intrathecal injection with NK antagonists or saline. No. 80.
128
O. LoÈfgren et al. / Burns 25 (1999) 125±129
Fig. 3. Changes in hindpaw withdrawal latency (HWLs) to pressure after intrathecal injection of NK antagonists and saline in scalded rats. No. 80.
treated control group, the increase was signi®cant in the group of rats treated with the NK1 antagonist ( p = 0.039). Treatment with the NK2 antagonist was followed by a less pronounced increase of HWL ( p = 0.58). But after the injection of NK1 nonpeptidergic antagonist the increase of HWL was signi®cant ( p = 0.007). The eect of NK1 peptidergic and nonpeptidergic antagonist on HWLs was similar ( p = 0.97). In all groups treated with tachykinin antagonists, there was a tendency towards increases in HWLs on the contralateral side. (Fig. 3).
5. Discussion The results of the present studies show that the administration of antagonists directed towards receptors of the sensory neuropeptides SP (NK1 receptor antagonist) and NKA (NK2 receptor antagonist) in¯uence thermally induced in¯ammation. Unilateral scalding of the hindpaw was followed by a progressive ipsilateral edema formation which was signi®cantly reduced by local treatment with the NK1 antagonist. Administration of the NK2 antagonist also reduced the edema formation, although to a lesser extent. After scalding there was a bilateral decrease in hindpaw withdrawal latency to mechanical stimulation with the decrease more pronounced on the thermally injured paw. Intrathecal administration of the NK1 antagonist as well as the nonpeptidergic NK1 antagonist signi®cantly increased the withdrawal responses. The localized edema reaction after thermal injury is initiated by an early fall in interstitial hydrostatic pressure followed by transcapillary ¯uid shift [9] and the release of dierent in¯ammatory mediators [10]. In previous studies it has been reported that the sensory nervous system contributes to in¯ammatory processes, and collaborators have reported that SP is released
locally after scalding [11]. We have recently reported that another tachykinin, NKA, is released into the subcutaneous space of the paw after scalding [2]. Pretreatment with the neurotoxin capsaicin or with a substance P antagonist decreases plasma extravasation after scalding [12, 13]. In other studies, using an online laser Doppler technique, we reported a biphasic perfusion increase during the initial phase of the edema formation [14] which can be signi®cantly reduced by pretreatment with NK1 and NK2 antagonists [15]. This is in accordance with the present ®nding where the NK1 antagonist signi®cantly reduced the edema formation after thermal injury and the NK2 antagonist had a similar but less pronounced eect. Thermal injury results in the activation of primary nociceptive aerents followed by the release of SP and NKA at the dorsal horn of the spinal cord [4]. Duggan et al. suggested that NKA is especially involved in the transmission of noxious thermal stimuli [16]. In present studies intrathecally administered NK1 or NK2 peptidergic receptor antagonists resulted in an increase of hindpaw withdrawal responses, an eect most apparent after treatment with NK1 antagonists. Since there are drawbacks in the use of receptor antagonists of peptidergic origin, dierent nonpeptidergic antagonists have been developed during the last decade [6]. Therefore it was decided also to investigate the eect of a nonpeptidergic NK1 receptor antagonist in the present study. The result was a signi®cant increase in hindpaw withdrawal latency, equal in eect to the peptidergic one. As described above, unilateral scalding to the hindpaw resulted in an ipsilateral edema formation and a bilateral decrease in hindpaw withdrawal latencies to mechanical pressure. This is in line with other results, indicating a communication of sensory activity from one side to the other [17, 18]. As earlier mentioned it has been proposed that primary sensory neurons
O. LoÈfgren et al. / Burns 25 (1999) 125±129
contributes both to the local in¯ammatory reaction and the transmission of nociceptive information centrally [19]. The neurogenic contribution to the peripheral in¯ammatory reaction has been attributed to local axon re¯exes and dorsal root re¯exes, resulting in the release of sensory neuropeptides [20]. Recently it has also been shown that excessive nociceptive activation at the central terminals of primary aerents results in an interneuronal circuitry which may be followed by increased activity in contralateral nociceptive terminals [20]. Taken together, the results show that unilateral thermal injury causes an unilateral edema formation which is a part of a local defense reaction, whereas the bilateral increase of withdrawal latency is a sign of a general hyperalgesic response. Acknowledgements The study was supported by funds from the AnnaGreta Crafoord's Foundation, the Gustav Vth 80-year Anniversary Foundation, the Karolinska Institutet Foundation, the Magnus Bergwall Foundation, the Nanna Swartz Foundation and the Swedish Society against Rheumatism (RMR). References [1] Yonehara N, Shibutani T, Inoki R. Contribution of substance P to heat-induced edema in rat paw. J Pharmacol Exp Ther 1987;242:1071±6. [2] LoÈfgren O, Palmer B, Theodorsson E, ToÈrkvist L, Lundeberg T. Contribution of the sensory and sympathetic nervous system to scalding-induced edema in the rat paw. Burns 1998;24:188±95. [3] Siney L, Brain SD. Involvement of sensory neuropeptides in the development of plasma extravasation in rat dorsal skin following thermal injury. Br J Pharmacol 1996;117:1065±70. [4] Levine JD, Fields HL, Basbaum AI. Peptides and the primary aerent nociceptor. J Neurosci 1993;13:2273±86. [5] Wiesenfeld-Hallin Z, Xu XJ. The dierential roles of substance P and neurokinin A in spinal cord hyperexcitability and neurogenic in¯ammation. Regul Peptides 1993;46:165±73.
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