The neurokinin-1 (NK-1) receptor and its ligand, substance P, are thought to play important roles in nociception and hyperalgesia. This study evaluated the role of the NK-1 receptor in processing noxious stimuli in normal and inflammatory states.
Behavioral responses to heat and mechanical and chemical stimuli were studied in NK-1 receptor knockout mice and wild-type control mice. Thermal nociception was evaluated by measuring paw lick or jump latencies to hot plate (52, 55, and 58 degrees C) and paw withdrawal latencies to radiant heat applied to the hind paws. Mechanical nociception was measured by von Frey monofilament applications to the hind paws. Intraplantar capsaicin-induced (10 microg/20 microl) paw licking and mechanical and heat hyperalgesia were compared in NK-1 knockout and wild-type mice.
Withdrawal responses to radiant heat (4.3+/-0.18 s for knockout and 4.4+/-0.8 s for wild-type mice) and von Frey monofilaments were similar in knockout and wild-type mice. In the hot plate test, increasing the hot plate temperature from 52 degrees C to 58 degrees C resulted in a decrease in the response latency in the wild-type mice (30.4+/-17.5 s to 15.2+/-6.8 s, P < 0.05), whereas in the knockout mice the response latencies remained constant (28.2+/-19.8 s to 29+/-15.1 s, not significant). Capsaicin-induced paw licking (14.5+/-12.8 s for knockout and 41.3+/-37.3 s for wild-type mice, P < 0.05) and mechanical and heat hyperalgesia were attenuated in the knockout mice.
NK-1 receptors contribute to the withdrawal responses to high-intensity heat stimuli and to capsaicin-induced mechanical and heat hyperalgesia.
Key words: Neurogenic inflammation; substance P; pain.
THE neurokinin-1 (NK-1) receptor and its ligand, substance P, are thought to play important roles in coding noxious stimuli and in the mechanisms of hyperalgesia. [1-4]Substance P is synthesized and released after noxious stimulation from the small-diameter primary afferents to the spinal cord dorsal horn. [5-8]Neurokinin-1 receptors are located in the superficial laminae of the dorsal horn of the spinal cord, a region thought to be important in the processing of nociceptive stimuli. Although substance P can excite spinal nociceptive neurons, its primary role is believed to be modulation of the responses of excitatory amino acids. Recent studies in mice with deletion of the gene encoding for substance P-neurokinin A (NKA) or the NK-1 receptor show alterations in pain sensitivity. A reduction in nociceptive responses to certain somatic and visceral noxious stimuli has been shown in substance P-NKA and NK-1 knockout mice. [12-14]In particular, the intensity coding of suprathreshold stimuli is impaired in the NK-1 knockout mice. Inflammation-induced mechanical hyperalgesia was similar between mutant and wild-type in substance P-NKA and NK-1 knockout mice. Mantyh et al. showed in rats that destroying the lamina I spinal cord neurons expressing the substance P receptor attenuates neurogenic inflammation-induced hyperalgesia. However, neurons expressing the substance P receptor also might express some other neurotransmitter receptors that play a role in the mechanisms of hyperalgesia. To characterize further the role of NK-1 receptors in nociception, we compared the behavioral responses to thermal, mechanical, and chemical stimuli applied to the normal and inflamed paws of NK-1 knockout and wild-type control mice.
Materials and Methods
The experiments were performed using NK-1 knockout (n = 22) and wild-type balb C (n = 21) adult mice (8-16 weeks old in both groups). Mice deficient in the NK-1 receptor were generated by gene targeting as described in another publication. Breeding stocks of NK-1 knockout mice and wild-type controls were provided by Dr. Norma Gerard (Perlmutter Laboratory, Children's Hospital, Boston, Massachusetts). Colonies were established in our laboratories and maintained in microisolator cages. The genomic status was monitored periodically by polymerase chain reaction. The animals were maintained in a 12-h light-dark cycle and were provided food and water ad libitum. The NK-1 receptor knockout animals were grossly normal developmentally, were fertile, and appeared to be healthy. Knockout animals could not be separated from the wild-type mice by their appearance. Locomotor activity has been shown to be normal in NK-1 knockout mice. For all the behavioral experiments, the animals were allowed to habituate for 5 days to the testing environment and for 15 min each day before the actual testing was started. All the testing was performed blinded to the genotype of the mice. The research protocol was approved by the Johns Hopkins Animal Care and Use Committee.
Hot Plate Test
In the hot plate test (wild-type, n = 12; NK-1 knockout, n = 10), mice were placed on a metal plate surrounded by a clear plastic chamber. The latency to lick one of the hind paws or to jump off the plate was measured. Paw lick or jump latencies were measured at three different temperatures. The sequence of hot plate temperatures was 55, 58, and 52[micro sign]C (wild-type, n = 10; NK-1 knockout, n = 6). For the remaining mice (wild-type, n = 2; NK-1 knockout, n = 4), the sequence was 58, 55, and 52[degree sign]C. In each animal, the response to only one temperature was tested on a given day. The tests were separated by at least 24 h. A cutoff time of 60 s was used.
To measure withdrawal latency to radiant heat, the mice (wild-type, n = 10; NK-1 knockout, n = 10) were placed on a glass plate preheated to a constant temperature surrounded by a clear plastic chamber (model 336 Analgesia Meter; IITC Inc./Life Science Instruments, Woodland Hills, CA). Before withdrawal latencies were measured, plantar surfaces of both hind paws were painted black with waterproof ink (Eberhard Faber, Lewisburg, TN). Radiant heat was applied from below to the plantar surface of each hind paw, and the withdrawal latency was measured using an electronic timer. Three measurements were taken in each hind paw spaced at least 1 min apart to determine the mean withdrawal latency.
Withdrawal Frequencies to Mechanical Stimuli
Mechanical withdrawal frequencies were measured by calibrated von Frey monofilaments (Stoelting Co., Wood Dale, IL). Mice (wild-type, n = 10; NK-1 knockout, n = 10) were placed in a clear plastic chamber on an elevated mesh screen. The von Frey monofilaments were applied to the plantar surface of each hind paw in ascending series. The monofilaments used in this study produced forces of 0.06, 0.20, 0.52, 1.15, 2.86, and 4.43 g. Every monofilament was applied to the hind paw for approximately 1 s. Each hind paw was studied with ascending series of monofilaments five times. The frequency percentage of paw withdrawal was calculated to correspond to each monofilament.
Capsaicin-induced Pain Behavior
To study the responses to chemical stimulus in wild-type and NK-1 knockout mice, we injected 10 [micro sign]g/20 [micro sign]l capsaicin to the intraplantar surface of the hind paw (wild-type, n = 14; NK-1 knockout, n = 13). Capsaicin was dissolved in 7.5% dimethylsulfoxide in saline. Vehicle injections (20 [micro sign]l) served as control for capsaicin injections (wild-type, n = 6; NK-1 knockout, n = 7). The licking time of the injected hind paw was measured for 10 min after injection. After capsaicin or vehicle injections, withdrawal latencies to radiant heat and mechanical withdrawal frequencies were determined in the same mice as described previously at various time points. Latencies to radiant heat were tested before (baseline) and 10, 40, and 60 min after injections. Mechanical testing was performed before and 10, 25, 45, and 65 min after injections.
Animals were tested on the hot plate test set at three different temperatures on separate days. Baseline mechanical and radiant heat paw withdrawal measurements were taken and were followed by the capsaicin or vehicle injections. After capsaicin or vehicle injections, mechanical and thermal testing were performed at various times for as long as 65 min after injection.
Data were analyzed using a two- or three-way repeated measures analysis of variance. Post hoc comparisons were performed using the Tukey significant difference test. All the statistical analyses were performed using the statistical software package Statistica (StatSoft, Tulsa, OK). P < 0.05 was considered significant.
Responses to Thermal and Mechanical Stimulation
The response latencies to the hot place test at 52[degree sign]C were similar in the wild-type and NK-1 knockout mice. Significant interaction existed between the group and the hot plate temperature (F2,40 = 4.79, P < 0.05). Increasing the hot plate temperature from 52[degree sign]C to 58[degree sign]C resulted in a decrease in the response latency in the wild-type mice (30.4 +/- 17.5 s to 15.2 +/- 6.8 s, P < 0.05; Figure 1A). In the NK-1 knockout mice, the response latencies remained constant over the increased temperature (28.2 +/- 19.8 s to 29 +/- 15.1 s, not significant; Figure 1A).
Withdrawal latencies to radiant heat were not different in the NK-1 knockout and wild-type mice in the uninflamed paw. The latencies to withdrawal were measured in both hind paws, and the latencies were averaged in each animal. In NK-1 mice, the withdrawal latency was 4.3 +/- 0.8 s; in wild-type mice, the latency was 4.4 +/- 0.8 s (Figure 1B).
The baseline withdrawal frequencies to mechanical stimulation of NK-1 knockout and wild-type mice were not significantly different in the uninflamed paw (F7,348 = 1.3, P > 0.05; Figure 2).
Capsaicin-induced Pain Behavior
Paw licking time after capsaicin injection in wild-type mice was longer than in the NK-1 knockout mice (41.3 +/- 37.3 s vs. 14.5 +/- 12.8 s, P < 0.05; Figure 3). The licking time after vehicle or capsaicin injections in the NK-1 knockout mice were not significantly different (10.5 +/- 10.4 s vs. 14.5 +/- 12.8 s, not significant; Figure 3).
There was a significant interaction between group and time (F21,168 = 1.84, P < 0.05) in the withdrawal latencies to radiant heat after capsaicin or vehicle injections. In the wild-type mice, 10 min after capsaicin injection, withdrawal latencies in the injected paw decreased (pre- vs. postinjection latencies: 4.4 +/- 0.8 s vs. 3.2 +/- 1.2 s, P < 0.01; Figure 4) and returned to baseline values 40 min after capsaicin injection (3.8 +/- 1.2 s, not significant; Figure 4). In the NK-1 knockout mice, capsaicin and vehicle injections did not decrease withdrawal latencies at any time (Figure 4). None of the injections had significant effects on the withdrawal latencies of the contralateral paw in wild-type or NK-1 knockout mice (F3,28 = 1.16, P > 0.05; data not shown).
After injection of capsaicin or vehicle, significant differences occurred in the mechanical withdrawal frequencies between NK-1 and wild-type mice. We found a main effect for the group (F7,252 = 11.07, P < 0.001), force (F5,252 = 391.63, P < 0.001), and time (F4,1008 = 14.82, P < 0.001). A significant interaction also occurred between group and time (F28,1008 = 9.14, P < 0.001). Capsaicin injection in the wild-type mice significantly increased the paw withdrawal frequencies compared with preinjection frequencies (Figure 5, A and B). The increased responsiveness seen 10 min after capsaicin injection returned to baseline 25 min after injection (Figure 5C). In the NK-1 knockout mice, capsaicin-induced mechanical hyperalgesia was attenuated. Vehicle injections did not alter withdrawal frequencies in NK-1 knockout or wild-type mice (Figure 5A, Figure 5B, Figure 5C). In addition, neither the capsaicin nor the vehicle injections to hind paws affected the withdrawal frequencies of the contralateral hind paw of wild-type or NK-1 knockout mice (F3,126 = 2.5, P > 0.05; data not shown).
We made three primary observations in the current study: (1) Nociceptive responses to mechanical and radiant heat stimuli in the normal paw were not different for NK-1 receptor knockout and wild-type mice; (2) in the NK-1 receptor knockout mice, the responses to high-intensity heat stimuli are attenuated, as indicated by the long response latencies to the high temperatures in the hot plate test; and (3) capsaicin-induced pain behavior and mechanical and thermal hyperalgesia are attenuated in the NK-1 receptor knockout mice.
Responses to Thermal Stimuli
Withdrawal responses to radiant heat were similar between mutant and wild-type mice. Our results are consistent with those of previous studies that used substance P-NKA or NK-1 receptor knockout mice. [12-14]Conflicting evidence has been reported for the response of the substance P-NKA knockout mice to different intensities of heat stimuli. Cao et al. showed that with moderate-intensity radiant heat stimuli to the paw, pain behavior is attenuated in substance P-NKA knockout mice, whereas Zimmer et al. failed to observe differences in tail flick responses to various intensities of radiant heat stimuli.
In the current study, the response latencies in the hot plate test remained constant as the temperatures increased in the NK-1 receptor knockout mice. In contrast, the response latencies decreased significantly in the wild-type mice. This result corresponds with findings of previous studies [12,13]that used substance P-NKA knockout mice and showed attenuation in the hot plate latencies in the knockout mice. It also has been shown that noxious heat stimuli to the skin results in an intensity-dependent release of substance P and NK-1 receptor internalization in the spinal cord. [8,19]However, De Felipe et al. did not find any differences between NK-1 knockout and wild-type mice in hot plate latencies at 52[degree sign]C. In the current study, response latencies at 52[degree sign]C also were not different between NK-1 knockout and wild-type mice. Results from current and previous studies indicate that substance P-NK-1 receptors contribute to the responses to high-intensity heat stimuli. Previous electrophysiologic and behavioral studies have shown that nociceptive responses to high and low rates of noxious cutaneous heating are mediated by different classes of nociceptors. Rapid heating of the skin preferentially activates A [Greek small letter delta] nociceptors, whereas a slower rate of heating preferentially activates C-fiber nociceptors. [20,21]It is possible that the NK-1 receptor is preferentially involved in the A [Greek small letter delta]-fiber-mediated nociception during physiologic conditions.
Responses to Mechanical Stimuli
Withdrawal responses to mechanical stimuli were similar in NK-1 and wild-type mice. This is in line with previous studies using substance P-NKA or NK-1 receptor knockout mice. Cao et al. showed, however, that responses to more intense mechanical stimuli were attenuated in mutant mice, indicating that tachykinins, substance P, and NKA play an important role in coding suprathreshold stimuli. Further evidence to support this view is derived from studies showing that high-frequency primary afferent stimulation or suprathreshold mechanical stimuli are needed to evoke a release of substance P. [22,23]In addition, De Felipe et al. did not see any intensity encoding to suprathreshold mechanical stimuli in NK-1 knockout mice. In the current study, withdrawal responses to suprathreshold von Frey monofilaments were not different between NK-1 knockout and wild-type mice. The mechanical stimuli used in these studies were more intense (for example, hemostat applied to the hind paw or a clip applied to the tail). Therefore, the intensity of mechanical stimuli in the current study might have been too weak to produce differences in responses to suprathreshold stimuli. Punctate stimuli delivered by von Frey monofilaments are short lasting and confined to a small area. In addition to the intensity of mechanical stimuli, temporal and spatial summation also may have contributed to the differences in responses to different mechanical stimuli.
Response to Capsaicin
Capsaicin has been shown to evoke spontaneous pain and thermal and mechanical hyperalgesia in humans and in animals. [24-26]We observed that capsaicin-induced pain behavior and mechanical and thermal stimuli-evoked hyperalgesia were attenuated in the NK-1 knockout mice.
The finding that capsaicin-induced paw licking was abolished in the NK-1 knockout mice is in line with previous studies, which showed that (1) capsaicin evokes a release of substance P in the spinal cord, (2) intrathecal substance P elicits pain behaviour, (3) capsaicin-induced pain behaviour can be attenuated by intrathecal administration of NK-1 or tachykinin antagonists, [26,28]and (4) capsaicin-induced pain behavior also was reduced in substance P-NKA knockout mice. 
Capsaicin-induced heat hyperalgesia was abolished in the NK-1 knockout mice. Mantyh et al. also reported similar attenuation of capsaicin-induced thermal hyperalgesia in rats with cytotoxic injury to lamina I NK-1 receptor-expressing neurons. These neurons also may express other neurotransmitter receptors that may play an important role in thermal hyperalgesia. Thus, the specific role of NK-1 receptors is difficult to assess from this study.
Substance P, released in the skin by noxious stimulation of primary afferents or by cutaneous injection of capsaicin, and NK-1 receptors have been shown to contribute to neurogenic inflammation. [12,14,29,30]In substance P-NKA or NK-1 receptor knockout mice, capsaicin-induced plasma extravasation and edema are reduced. Amann et al. showed that the systemic administration of NK-1 receptor antagonist SR140333 inhibits neurogenic inflammation at a dose with no effect on acute chemoception or thermonociception in rats. Results from the electrophysiologic experiments indicate that substance P does not excite nociceptors. However, peripherally administered substance P can sensitize nociceptors to other inflammatory mediators, thus contributing to the pain and thermal hyperalgesia in inflammation. These studies suggest that the attenuation of neurogenic inflammation induced pain behavior and hyperalgesia in NK-1 receptor knockout animals is not merely the result of a lack of an inflammatory response in the periphery.
We observed that capsaicin-induced mechanical hyperalgesia is attenuated in the NK-1 knockout mice. This observation is consistent with the report by Mantyh et al. that indicates the role of NK-1 receptor-expressing neurons in capsaicin-induced mechanical hyperalgesia. The authors showed that capsaicin-induced mechanical hyperalgesia was inhibited in rats given a substance P-saporin toxin that selectively ablated the NK-1 receptor-expressing neurons in lamina I of the spinal cord. The contribution of substance P and NK-1 receptors in other experimental models of pain leading to mechanical and thermal hyperalgesia also has been reported [2,4,33,34]These data obtained from studies in receptor knockout animals should be interpreted with caution because compensatory changes may occur during the animal's development in response to the lack of receptors that are normally present.
The data from the current study indicate that NK-1 receptors contribute to the responses to high-intensity heat and chemical stimuli. NK-1 receptors do not play a major role in responses to noxious stimuli near the nociceptive threshold. However, in neurogenic inflammation, NK-1 receptors contribute to heat and mechanical hyperalgesia.
The authors thank Tim Hartke and Camille Dickerson for technical assistance and Dr. Norma P. Gerard for providing breeding stocks of NK-1 receptor knockout mice for in-house breeding.