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The algogenic-induced nociceptive flexion test in mice: studies on sensitivity of the test and stress on animals
Brain Research Bulletin 60 (2003) 275–281
The algogenic-induced nociceptive flexion test in mice: studies on sensitivity of the test and stress on animals Makoto Inoue a , Md Harunor Rashid a , Toshiko Kawashima a , Misaki Matsumoto a , Takehiko Maeda b , Shiroh Kishioka b , Hiroshi Ueda a,∗ a
Division of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan b Department of Pharmacology, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-0012, Japan Received 19 September 2002; received in revised form 22 January 2003; accepted 22 January 2003
Abstract Recently we developed a new technique, known as peripheral nociception test or algogenic-induced nociceptive flexion (ANF) test, to study the in vivo signal transduction of pain at the peripheral nerve endings in mice. In the present report, we examined the sensitivity of the method to detect pain signal and the stresses induced by the test on experimental animals. In the algogenic-induced biting and licking (ABL) test, bradykinin could not induce significant biting–licking response even at a dose of 1 nmol. It induced significant biting–licking response only at 10 nmol. However, with the ANF test, 100 fmol of bradykinin was enough to produce sharp and significant nociceptive flexion response. Similarly, substance P, ATP and ONO-54918-07, a stable prostaglandin I2 agonist, induced nociceptive flexion response in ANF test at much lower doses than needed to induce biting–licking responses in ABL test. Next, we measured the plasma corticosterone level after different nociception tests, which is a measure of stress on animals due to experimental manipulations. However, no significant rise in corticosterone level was observed with ANF test. Altogether, these findings indicate that the ANF test is a highly sensitive and less stressful technique to study in vivo mechanisms of pain at the peripheral nerve ending. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Nociceptors; Peripheral; Algogenic; Pain; Stress; Plasma corticosterone
1. Introduction Our understandings of the mechanism of nociception are largely based on experimental animal models. There are numerous models of nociception in animals, mainly the rodents [9]. Most of these behavioral models of nociception measure the output responses induced by various input stimuli. The most common input stimuli are electrical, thermal, mechanical and chemical. Although all of these stimuli have many advantages, they also have some drawbacks. In addition to direct activation of the mechanothermal A␦- and polymodal C-fiber nociceptors, these stimuli are expected to drive multiple indirect mechanisms including release of pain-producing substances such as bradykinin (BK), histamine, ATP and potassium ions [9].
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Corresponding author. Tel.: +81-95-844-4277; fax: +81-95-844-4248. E-mail address: [email protected] (H. Ueda).
Very recently, we have developed a novel technique which measures the nociceptive flexion response induced by extremely low doses of different algogenics at the peripheral nerve endings in mice [19,22]. This algogenic-induced nociceptive flexion (ANF) test has been advantageous for the study of in vivo signal transduction of pain at the peripheral nerve ending [5,20]. Using this novel technique of ANF test in na¨ıve mice, recently we have hypothesized for three distinct types of nociceptive fibers depending on their sensitivity to specific peripheral receptor ligand stimulus [22]. The nociceptors called neonatal capsaicin-sensitive type I, stimulated by intraplantar injection (i.pl.) of substance P or BK were characterized by their sensitivity to neonatal capsaicin treatment and intrathecal (i.t.) neurokinin 1 (NK1) receptor antagonist [5,14,20,22]. This type corresponds to the well-known class I polymodal C fibers reported by Snider and McMahon [16], since they share the substance P neurotransmission at the level of spinal cord. The nociceptors called neonatal capsaicin-sensitive
0361-9230/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0361-9230(03)00045-5
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type II, stimulated by purinergic P2X3 receptor agonists were characterized by their sensitivity to neonatal capsaicin treatment and i.t. N-methyl-d-aspartate (NMDA) receptor antagonist, but not the i.t. NK1 receptor antagonist. We have also shown the presence of neonatal capsaicininsensitive nociceptors (named as type III), stimulated by prostaglandin I2 (PGI2 ) agonist and characterized by neonatal capsaicin-insensitivity and i.t. NMDA receptor antagonist-sensitivity [22]. The pain transmitting sensory system is a complex mechanism consisting of the external stimuli and the internal milieu of the organism. Moreover, the sensory discriminatory and motivational affective components of pain are also likely to overlap to some extent to the neural pathways that mediate normal pain sensation. Thus, psychological factors like distraction, fear and other environmental stresses modulate the way of pain perception [3]. Consequently, the stresses induced by various manipulations in different nociception tests need to be duly addressed. Unfortunately there had been no such reports until now. In the present report, we first examined the sensitivity of the ANF test to induce nociceptive responses at the peripheral nerve ending. Next, we studied the effects of experimental manipulations to produce stresses on experimental animals during the ANF test as well the other conventional nociception tests.
2. Materials and methods All procedures throughout the present study were approved by Nagasaki University Animal Care Committee. All experiments were performed in compliance with the relevant laws and institutional guidelines including the U.K. Animals Act, 1986 and European Communities Council Directive, 1986, and complied with the ethical guidelines of the International Association for the Study of Pain [25]. In all circumstances, maximum possible efforts had been made to minimize animal sufferings and to reduce the number of animals used in the experiments. 2.1. Experimental animals Male ddY mice weighing 20–25 g were used throughout the experiments. They were housed in the animal facility of the University which had been always maintained at 21 ± 2 ◦ C, 55 ± 5% relative humidity and an automatic 12 h light–dark cycle. The animals were kept in a group of six and received standard laboratory diet (Oriental Yeast Co. Ltd., Japan) and tap water ad libitum. The animals were adapted to the testing environment (maintained at 21 ± 2 ◦ C, 55 ± 5% relative humidity and 12 h light–dark cycle) by keeping them in the testing room 24 h before the experiments. Experiments were performed during the light phase of the cycle (10:00–17:00 h).
2.2. Drugs The following drugs were used: bradykinin (BK; Sigma, St. Louis, MO, USA), substance P (SP; Peptide Institute Osaka, Japan), adenosine triphosphate (ATP; Research Biochemicals International, MA, USA), formalin (Nacalai Tesque, Japan). The stable PGI2 agonist, ONO-54918-07 [15-cis-(4-n-propylcyclohexyl)-16,17,18,19,20-pentanor-9deoxy-6,9-alpha-nitriloprostaglandin F1] was kindly provided by Ono Pharmaceutical Co. Ltd., Osaka, Japan. All drugs were dissolved in physiological saline (pH 7.4). The pH of the drug solutions did not differ from the vehicle pH (7.4). 2.3. Algogenic-induced nociceptive flexion (ANF) test The ANF test or the peripheral nociception test had been performed as described previously [1,5,19,21,22]. The nociceptive flexion response induced by i.pl. injection of a single molecule of receptor ligand or algogenic was measured. Mice weighing usually between 20 to 22 g were used for this test. Na¨ıve mice were first lightly anaesthetized with ether and held in a cloth sling with their four limbs hanging free through holes. The sling was suspended on a metal bar. All limbs were tied with soft thread strings. Then three limbs were fixed to the floor, while the other one (right hind-limb) was connected to an isotonic transducer and recorder. All experiments were started after complete recovery from the light ether anesthesia. A polyethylene cannula (0.61 mm in outer diameter) filled with specific algogenic substance was connected to a 50-l Hamilton microsyringe and then carefully inserted into the undersurface of the right hind-paw via a 30-gauge hypodermic needle. The pain due to insertion of the needle caused some immediate non-specific flexion responses recorded as the peak height in centimeters over the recording sheet. The biggest response among these non-specific flexion responses occurred immediately following cannulation was considered as the maximal reflex. This maximal reflex is considered as the maximum possible flexion capacity of the animal. The nociceptive flexion responses induced by different algogenic substances infused in a volume of 2 l were then measured. About four to five challenges with the algogenic were made in each mouse at a time interval of 5 min. The flexion responses induced by various algogenics were represented as the percentage of maximal reflex in each mouse as the flexion forces differ from mouse to mouse. The control response was taken as the average of the three consecutive vehicle-induced responses in the beginning of each experiment. 2.4. Algogenic-induced biting and licking (ABL) test The algogenic-induced biting and licking (ABL) test was performed as described previously [18]. In the ABL test, the nociceptive biting and licking responses induced
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by i.pl. injection of different algogenic substances were measured. Mice were placed in a Plexiglas cage for 1 h to adapt to the environment. Before the test, mice were restrained in hand and gently taken inside a hard paper tube of internal diameter 2.5 cm. The right hind-paw was taken out of the tube and algogenic substances (BK, SP, ATP or ONO-54918-07) were injected into the paw in a volume of 20 l using a 30-gauge needle fitted to a Hamilton microsyringe. Mice were immediately put back to the cage and the time spent on biting and licking of the injected paw was measured with stopwatch for a period of 10 min after injection. Saline was injected as control. Mice were used only once. 2.5. Different conventional nociception tests In Hargreaves thermal paw withdrawal test, paw withdrawal latency was measured by exposing the right hind-paw to a thermal stimulus [4]. Unanaesthetized animals were placed in Plexiglas cages on top of a glass sheet and an adaptation period of 1 h was allowed. The thermal stimulus was positioned under the glass sheet to focus the projection bulb exactly on the middle of plantar surface of the animals and the latency to withdrawal of the paw was measured. Test was performed for 30 min at 10-min interval with a thermal latency of ∼10 s in na¨ıve mice. Paw pressure test was performed as described previously [1,13]. Mice were placed into a Plexiglas chamber on a 6 mm × 6 mm wire mesh grid floor and were allowed to accommodate for a period of 1 h. The mechanical stimulus was then delivered onto the middle of the plantar surface of the right hind-paw using a Transducer Indicator, and the withdrawal threshold was measured. Paw pressure test was performed for 30 min at 10-min interval with a pressure threshold of ∼10 g in na¨ıve mice. In the tail-flick test, animals were gently restrained by hand, and radiant heat was focused onto the marked black ventral surface of the tail as described previously [21]. The tail-flick response was evaluated for 30 min at 10-min interval with a thermal latency of ∼10 s in na¨ıve mice. In the formalin test, a 20-l aliquot of 1% formalin solution was administered into the hind-paw of na¨ıve mice with a Hamilton microsyringe, and the induced behaviors such as licking and biting of the injected paw were evaluated for 30 min as nociceptive responses [23]. 2.6. Measurement of plasma corticosterone level The plasma corticosterone level was measured as described previously with little modifications [7]. All nociception tests including the ANF test were performed for a period of 30 min. The mice were decapitated immediately after each nociception test and the whole blood was collected. The time interval between the noxious stimulus and manipulations until sacrifice were strictly maintained similar (1 min) among the different nociception tests. The
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plasma was separated by centrifuging at 3000×g for 15 min at 4 ◦ C. It was then collected into ice-chilled tubes containing 0.1% EDTA and stored at −80 ◦ C until assayed. The blood sample was collected between 10:00 and 11:00 h in order to exclude the effect of a circadian rhythm on circulating plasma corticosterone. The plasma coticosterone level was estimated fluorometrically according to the method of Zenker and Bernstein [24]. 2.7. Statistical analysis Results in the ANF test were analyzed with one-way analysis of variance and Dunnett’s post hoc test. Results in the ABL test were analyzed with one-way analysis of variance and Sheffe’s post hoc test. Statistical analysis of the plasma corticosterone data was made using the Student’s t-test. All data were presented as mean ± S.E.M. P values less than 0.05 were considered to indicate statistical significance.
3. Results 3.1. The ANF test is more sensitive to induce nociceptive response compared to the ABL test Fig. 1A and B shows the diagrammatic model of ANF test in mice and some representative traces of BK-induced nociceptive flexion responses, respectively. As shown in Fig. 1A, the mouse was taken into a soft cloth sling and three limbs were gently tied with soft cotton thread and the fourth limb (right hind) was connected to the lever of the isotonic transducer which was connected to a recorder. As shown in Fig. 1B, the mouse gave some biggest non-specific flexion responses immediately following cannulation producing some highest peaks on the recording sheet. After an adaptation period of about 5–10 min, i.pl. injection of vehicle saline did not produce any flexion response. However, i.pl. injection of 2 pmol of BK produced very sharp and stable nociceptive flexion responses injected at every 5-min interval without any test–retest effect (Fig. 1B). We next performed experiments to compare the doseresponse pattern of various algogenic substances in the ANF and ABL tests. In the ANF test, the initial flexion response induced within seconds upon i.pl. infusion of the algogenics was evaluated by normalizing it with the maximal flexion response in each mouse. The maximal flexion response was the biggest response among the non-specific flexion responses which occurred immediately following cannulation (Fig. 1B). We consider it as the maximum possible flexion capacity of the animal. The result has been represented as the percentage of the maximal flexion response as the flexion forces differ from mouse to mouse. The control vehicle response for each mouse was taken as the average responses upon three consecutive saline injections. As shown in Fig. 2A, i.pl. injection of BK produced dose-dependent
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Fig. 1. Model of the algogenic-induced nociceptive flexion (ANF) test in mice. (A) Diagrammatic model of the ANF test in mice. The mice were taken in soft cloth sling and three limbs were gently tied with soft cotton thread and the fourth limb (right hind) was connected to the lever of the isotonic transducer which was then connected to a recorder. The nociceptive flexion response induced by various algogenic substances was quantified as height in centimeters on a paper chart. (B) A representative trace of the bradykinin-induced flexion responses. After cannulation at right hind-paw, the mouse gave some of the biggest and non-specific flexion responses which diminished within minutes. After an adaptation period of about 10 min, i.pl. injection of saline did not produce any flexion response. On the other hand, repeated challenge of bradykinin (2 pmol, i.pl.) at every 5 min produced very sharp and stable flexion responses.
Fig. 2. Comparison between the algogenic-induced nociceptive flexion (ANF) and algogenic-induced biting and licking (ABL) tests in mice. (A) Dose-response curves for the bradykinin (BK)-induced type I nociceptive flexion responses in the ANF test and biting–licking responses in the ABL test. (B) Substance P (SP)-induced type I nociceptive flexion response in the ANF test and the biting–licking behavior in the ABL test. (C) Dose-dependent ATP-induced type II flexion response and biting–licking behavior in the ANF and ABL tests respectively. (D) Dose-response curves for the PGI2 agonist, ONO-54918-07-induced type III responses in the ANF test and biting–licking responses in the ABL test. In the ANF test, results are presented as percentage of maximal reflex (see Section 2 for details). In the ABL test, time spent (in seconds) in biting and licking of the injected paw for a period of 10 min after injection was measured. All data points represents mean ± S.E.M. The vertical bars indicate the standard error of the means. Number of animals for each data point in the ANF test was nine. The number of animals for each data point in the ABL test is indicated in parenthesis. For the ANF test, the data were analyzed using one-way ANOVA and Dunnett’s test for post hoc comparisons. For the ABL test, the data were analyzed using one-way ANOVA and Scheffe’s test for post hoc comparisons. ‘Veh’ means vehicle saline data. (∗ ) Indicates significant difference compared with the vehicle saline response at a P value of less than 0.05.
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nociceptive flexion responses from 0.1 fmol to 10 pmol through stimulation of neonatal capsaicin-sensitive type I fibers in the ANF test with an ED50 of 188.5 ± 53.2 fmol. BK produced significant flexion responses at doses of 100 fmol, 1 and 10 pmol compared with the control saline response (1.98 ± 0.95%) [F(6, 56) = 36.06; P < 0.0001]. In the ABL test, on the other hand, the total biting–licking time (in seconds) over a period of 10 min upon i.pl. injection of the algogenics was measured. No significant struggling or vocalization of the animal was observed after i.pl. injection of the algogenics in the ABL test. In this nociception test, i.pl. injection of BK produced dose-dependent nociceptive biting–licking response from 1 pmol to 10 nmol. However, significant biting–licking response was observed only with 10 nmol of BK compared with the vehicle saline (0.95 ± 0.32 s versus 25.40 ± 6.09 s) [F(3, 21) = 7.59; P = 0.001]. Similar to BK, i.pl. injection of SP, another stimulant of type I fibers, induced dose-dependent and potent nociceptive flexion responses in the ANF test from doses of 1 fmol to 10 pmol with an ED50 of 165.3 ± 18.7 fmol (Fig. 2B). SP produced significant flexion responses in the ANF test at doses of 10 and 100 fmol, 1 and 10 pmol compared with the control saline response (1.10 ± 0.56%) [F(5, 48) = 49.23; P < 0.0001]. On the other hand, in ABL test no significant biting–licking responses could be observed even with 10 nmol of SP compared with the vehicle saline (Fig. 2B) [F(2, 17) = 2.02; P = 0.16]. ATP, the stimulant of neonatal capsaicin-sensitive type II fibers also induced dose-dependent flexion responses from 1 fmol to 100 pmol in the ANF test with an ED50 of 36.9 ± 5.3 pmol (Fig. 2C). Significant flexion response compared with the vehicle saline response (1.22 ± 0.94%) was observed at 100 fmol, 1, 10 and 100 pmol [F(6, 56) = 58.73; P < 0.0001]. ATP also produced dose-dependent biting–licking response in the ABL test from doses of 100 pmol to 10 nmol (Fig. 2C). Significant biting–licking response was observed only at 10 nmol compared with vehicle saline injection (0.71 ± 0.34 s versus 23.48 ± 4.16 s) [F(3, 24) = 11.47; P < 0.0001]. The PGI2 agonist, ONO-54918-07 induced neonatal capsaicin-insensitive type III nociceptive responses from doses of 1 fmol to 100 pmol in the ANF test with the ED50 of 8.7 ± 2.7 pmol (Fig. 2D). ONO-54918-07 produced significant flexion responses in the ANF test at doses of 1, 10 and 100 pmol compared with the control saline response (1.44 ± 0.73%) [F(6, 56) = 23.19; P < 0.0001]. However, as shown in Fig. 2D, no significant biting–licking response could be observed with even 10 nmol of ONO-54918-07 in the ABL test compared with the vehicle saline (0.95±0.32 s versus 0.95 ± 0.58 s) [F(2, 13) = 0.15; P = 0.86]. Statistical comparisons of the responses in the ANF and ABL tests could not be performed due to their different natures. However, from the figures it is clearly evident that the initial active dose of all algogenics was much lower in the ANF test than in the ABL test, indicating better sensitivity of the ANF test to produce nociceptive response at extremely low stimulation.
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Fig. 3. Plasma corticosterone level after different nociception tests. The plasma corticosterone was measured in na¨ıve mice and in mice after different nociception tests. Immobilization of mice in a 50-ml conical tube for 30 min was used as positive control. In formalin test, 1% formalin (in 20 l) was injected into the right hind-paw. In tail-flick test, radiant heat was applied into ventral surface of the tail. In Hargreaves test, radiant heat was applied into the paw. In paw pressure test, pressure was applied into the paw. The ANF test was performed as described in Section 2. In control ANF (ANF-control) test, only saline was injected. In the ANF test with bradykinin (ANF-BK), 2 pmol of BK was injected i.pl. All tests were performed for 30 min. In all cases, the blood sample for corticosterone measurement was collected immediately following the test with no difference in time interval among the tests. Each data point represents mean ± S.E.M. from 12 mice. The vertical bars indicate the standard error of the means. Data were analyzed using Student’s t-test. (∗ ) Indicates significant difference compared with the control mice at P < 0.05.
3.2. Effects of various nociception tests on the plasma corticosterone level Circulating plasma corticosterone level is used as a measure of stress on animal. We measured the plasma corticosterone level in mice after the ANF test as well as some conventional nociception tests. As shown in Fig. 3, plasma corticosterone in control na¨ıve mice was 12.3 ± 1.6 g/dl. Immobilization of the animal in a 50-ml conical tube for 30 min served as positive control which caused an abrupt rise in plasma corticosterone level (40.1 ± 2.4 g/dl). When the plasma corticosterone level was measured after the formalin test, significant increase in corticosterone level in the mice was observed compared with the control mice (12.3 ± 1.6 g/dl versus 29.7 ± 1.6 g/dl; Student’s t-test, P < 0.05). The thermal tail-flick test also caused significant rise in plasma corticosterone level in mice compared with the control mice (12.3 ± 1.6 g/dl versus 28.6 ± 1.3 g/dl; Student’s t-test, P < 0.05). However, there was no significant increase in the corticosterone level in mice after the ANF test (ANF-BK: 16.91 ± 2.7 mg/dl), Hargreaves thermal nociception test (17 ± 2.9 mg/dl) or the mechanical
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paw pressure test (17.5 ± 1.9 g/dl) compared with the control na¨ıve mice (Student’s t-test, P > 0.05). There was no effect of the slight ether anesthesia in the ANF test on the plasma corticosterone level since similar results were obtained without this initial slight anesthesia (data not shown).
4. Discussion An ideal model of nociception should conform to certain characteristics including specificity, sensitivity, validity and reliability. We have already established the ANF test as a stimulus-specific and reliable method to study pain mechanism at periphery [5,19,20,22]. The method is also sufficiently sensitive to induce nociception by local application of extremely low doses of pain-producing substances. In the present report, we have reconfirmed the highly sensitive nature of this test. In most conventional chemical nociception tests after cutaneous application of algogenics, chemical stimulation occurs slowly after administration of algogenic substances. Thus, in such tests, the response is usually measured as behavioral scores in units of time. The ABL test falls under this category of chemical nociception test. On the other hand, the ANF test measures the flexion reflex response induced by different doses of an algogenic substance and the response occurs within the fraction of a second. When we compared the dose-response curves of different algogenic substances by these two types of nociception tests, it was evident that the ANF test is much more sensitive than the ABL test (Fig. 2A–D). The i.pl. application of BK into rodent’s paw is reported to produce both thermal and mechanical hyperalgesia at lower doses [17]. In our present report, i.pl. injection of BK induced a dose-dependent biting–licking behavior in the mice from 1 pmol to 10 nmol (Fig. 2A). However, significant biting–licking response was observed only at a dose of 10 nmol of BK compared with the vehicle saline injection (Fig. 2A). On the other hand, i.pl. injection of BK produced very sharp nociceptive flexion response from doses of 0.1 fmol to 10 pmol in the ANF test which is consistent with our previous reports [6,22]. Thus, BK was about 10,000-times more potent in the ANF test than in the ABL test. In other words, the ANF test was about 10,000-times more sensitive to produce BK-induced nociception than the ABL test. Similarly, SP, ATP and PGI2 agonist also induced dose-dependent nociceptive flexion reflex responses in the ANF test as reported previously [5,21]. The doses of SP, ATP or PGI2 agonist that produced flexion response were much lower than the doses needed to produce biting–licking responses (Fig. 2B–D). Although direct statistical comparison of the responses in the ANF and ABL tests could not be performed due to their different natures, we attempted to compare the two tests with regard to the initial active dose of different algogenics. As shown in Fig. 2A–D, the initial active dose of all algogenics was much lower in the ANF
test than in the ABL test, indicating better sensitivity of the ANF test to produce nociceptive response at extremely low stimulation. Thus, the initial significantly active dose of BK was 100 fmol in the ANF test while it was 10 nmol in the ABL test (Fig. 2A). This highly sensitive nature of the ANF test is reflected by the fact that, the nociceptive responses in this test appeared to involve relatively simple molecular and neuronal mechanisms, in contrast to the ABL test which use strong and sustained chemical stimuli leading to the release of several endogenous pain-producing substances, such as BK, SP, somatostatin, histamine and glutamate [11,12]. Experimental manipulations may radically alter the outcome of any behavioral model of nociception. Psychological stresses produce analgesic effects in both human and animals [2,3,10]. Such stress-induced analgesia has been reported to be either opioid- or nonopioid-mediated. On the other hand, anxiety has been regarded as an aggravating factor for clinical pain. Indeed, the facilitatory effect of anxiety on the action of morphine on a pain threshold was identified long ago [8]. Thus, it has become important to assess the effects of different manipulations on the experimental animals during the nociceptive tests. Increase in circulating plasma coricosterone level is used as an indication of acute stress on animals [15]. In this context, we have measured the circulating plasma corticosterone level in mice after the ANF test as well the other conventional tests of nociception. Interestingly, significant increase in plasma corticosterone level was observed after the formalin and tail-flick tests compared to plasma corticosterone level in control animals (Fig. 3). On the other hand, although little rise in plasma corticosterone was observed after the ANF, Hargreaves thermal nociception or mechanical paw pressure tests, they were statistically insignificant compared to the control level (Fig. 3). The plasma corticosterone level in the ANF test after saline and BK injection were not also significantly different (Fig. 3, last two columns). The significant rise in corticosterone level with the tail-flick test might be due to direct handling of animal during the test. On the other hand, the persistent pain on animal in formalin test may underlie the significant increase in corticosterone level in this test. However, relatively little stress on animals in the Hargreaves thermal test, mechanical paw pressure test or the ANF test is understood by the fact that there is no direct contact between the animals and the experimenter in these tests. In conclusion, we demonstrate that the ANF test is highly sensitive to detect pain signal at extremely low doses of algogenic substances. The simplicity of the neuronal network at the peripheral nerve ending makes the test more specific and sensitive than other conventional tests. Moreover, our results of plasma corticosterone level for the first time revealed the amount of stress on animals in different nociception tests. The ANF test has been found to produce almost no stress in animals due to experimental manipulations.
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The algogenic-induced nociceptive flexion test in mice: studies on sensitivity of the test and stress on animals Makoto Inoue a , Md Harunor Rashid a , Toshiko Kawashima a , Misaki Matsumoto a , Takehiko Maeda b , Shiroh Kishioka b , Hiroshi Ueda a,∗ a
Division of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan b Department of Pharmacology, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-0012, Japan Received 19 September 2002; received in revised form 22 January 2003; accepted 22 January 2003
Abstract Recently we developed a new technique, known as peripheral nociception test or algogenic-induced nociceptive flexion (ANF) test, to study the in vivo signal transduction of pain at the peripheral nerve endings in mice. In the present report, we examined the sensitivity of the method to detect pain signal and the stresses induced by the test on experimental animals. In the algogenic-induced biting and licking (ABL) test, bradykinin could not induce significant biting–licking response even at a dose of 1 nmol. It induced significant biting–licking response only at 10 nmol. However, with the ANF test, 100 fmol of bradykinin was enough to produce sharp and significant nociceptive flexion response. Similarly, substance P, ATP and ONO-54918-07, a stable prostaglandin I2 agonist, induced nociceptive flexion response in ANF test at much lower doses than needed to induce biting–licking responses in ABL test. Next, we measured the plasma corticosterone level after different nociception tests, which is a measure of stress on animals due to experimental manipulations. However, no significant rise in corticosterone level was observed with ANF test. Altogether, these findings indicate that the ANF test is a highly sensitive and less stressful technique to study in vivo mechanisms of pain at the peripheral nerve ending. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Nociceptors; Peripheral; Algogenic; Pain; Stress; Plasma corticosterone
1. Introduction Our understandings of the mechanism of nociception are largely based on experimental animal models. There are numerous models of nociception in animals, mainly the rodents [9]. Most of these behavioral models of nociception measure the output responses induced by various input stimuli. The most common input stimuli are electrical, thermal, mechanical and chemical. Although all of these stimuli have many advantages, they also have some drawbacks. In addition to direct activation of the mechanothermal A␦- and polymodal C-fiber nociceptors, these stimuli are expected to drive multiple indirect mechanisms including release of pain-producing substances such as bradykinin (BK), histamine, ATP and potassium ions [9].
∗
Corresponding author. Tel.: +81-95-844-4277; fax: +81-95-844-4248. E-mail address: [email protected] (H. Ueda).
Very recently, we have developed a novel technique which measures the nociceptive flexion response induced by extremely low doses of different algogenics at the peripheral nerve endings in mice [19,22]. This algogenic-induced nociceptive flexion (ANF) test has been advantageous for the study of in vivo signal transduction of pain at the peripheral nerve ending [5,20]. Using this novel technique of ANF test in na¨ıve mice, recently we have hypothesized for three distinct types of nociceptive fibers depending on their sensitivity to specific peripheral receptor ligand stimulus [22]. The nociceptors called neonatal capsaicin-sensitive type I, stimulated by intraplantar injection (i.pl.) of substance P or BK were characterized by their sensitivity to neonatal capsaicin treatment and intrathecal (i.t.) neurokinin 1 (NK1) receptor antagonist [5,14,20,22]. This type corresponds to the well-known class I polymodal C fibers reported by Snider and McMahon [16], since they share the substance P neurotransmission at the level of spinal cord. The nociceptors called neonatal capsaicin-sensitive
0361-9230/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0361-9230(03)00045-5
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type II, stimulated by purinergic P2X3 receptor agonists were characterized by their sensitivity to neonatal capsaicin treatment and i.t. N-methyl-d-aspartate (NMDA) receptor antagonist, but not the i.t. NK1 receptor antagonist. We have also shown the presence of neonatal capsaicininsensitive nociceptors (named as type III), stimulated by prostaglandin I2 (PGI2 ) agonist and characterized by neonatal capsaicin-insensitivity and i.t. NMDA receptor antagonist-sensitivity [22]. The pain transmitting sensory system is a complex mechanism consisting of the external stimuli and the internal milieu of the organism. Moreover, the sensory discriminatory and motivational affective components of pain are also likely to overlap to some extent to the neural pathways that mediate normal pain sensation. Thus, psychological factors like distraction, fear and other environmental stresses modulate the way of pain perception [3]. Consequently, the stresses induced by various manipulations in different nociception tests need to be duly addressed. Unfortunately there had been no such reports until now. In the present report, we first examined the sensitivity of the ANF test to induce nociceptive responses at the peripheral nerve ending. Next, we studied the effects of experimental manipulations to produce stresses on experimental animals during the ANF test as well the other conventional nociception tests.
2. Materials and methods All procedures throughout the present study were approved by Nagasaki University Animal Care Committee. All experiments were performed in compliance with the relevant laws and institutional guidelines including the U.K. Animals Act, 1986 and European Communities Council Directive, 1986, and complied with the ethical guidelines of the International Association for the Study of Pain [25]. In all circumstances, maximum possible efforts had been made to minimize animal sufferings and to reduce the number of animals used in the experiments. 2.1. Experimental animals Male ddY mice weighing 20–25 g were used throughout the experiments. They were housed in the animal facility of the University which had been always maintained at 21 ± 2 ◦ C, 55 ± 5% relative humidity and an automatic 12 h light–dark cycle. The animals were kept in a group of six and received standard laboratory diet (Oriental Yeast Co. Ltd., Japan) and tap water ad libitum. The animals were adapted to the testing environment (maintained at 21 ± 2 ◦ C, 55 ± 5% relative humidity and 12 h light–dark cycle) by keeping them in the testing room 24 h before the experiments. Experiments were performed during the light phase of the cycle (10:00–17:00 h).
2.2. Drugs The following drugs were used: bradykinin (BK; Sigma, St. Louis, MO, USA), substance P (SP; Peptide Institute Osaka, Japan), adenosine triphosphate (ATP; Research Biochemicals International, MA, USA), formalin (Nacalai Tesque, Japan). The stable PGI2 agonist, ONO-54918-07 [15-cis-(4-n-propylcyclohexyl)-16,17,18,19,20-pentanor-9deoxy-6,9-alpha-nitriloprostaglandin F1] was kindly provided by Ono Pharmaceutical Co. Ltd., Osaka, Japan. All drugs were dissolved in physiological saline (pH 7.4). The pH of the drug solutions did not differ from the vehicle pH (7.4). 2.3. Algogenic-induced nociceptive flexion (ANF) test The ANF test or the peripheral nociception test had been performed as described previously [1,5,19,21,22]. The nociceptive flexion response induced by i.pl. injection of a single molecule of receptor ligand or algogenic was measured. Mice weighing usually between 20 to 22 g were used for this test. Na¨ıve mice were first lightly anaesthetized with ether and held in a cloth sling with their four limbs hanging free through holes. The sling was suspended on a metal bar. All limbs were tied with soft thread strings. Then three limbs were fixed to the floor, while the other one (right hind-limb) was connected to an isotonic transducer and recorder. All experiments were started after complete recovery from the light ether anesthesia. A polyethylene cannula (0.61 mm in outer diameter) filled with specific algogenic substance was connected to a 50-l Hamilton microsyringe and then carefully inserted into the undersurface of the right hind-paw via a 30-gauge hypodermic needle. The pain due to insertion of the needle caused some immediate non-specific flexion responses recorded as the peak height in centimeters over the recording sheet. The biggest response among these non-specific flexion responses occurred immediately following cannulation was considered as the maximal reflex. This maximal reflex is considered as the maximum possible flexion capacity of the animal. The nociceptive flexion responses induced by different algogenic substances infused in a volume of 2 l were then measured. About four to five challenges with the algogenic were made in each mouse at a time interval of 5 min. The flexion responses induced by various algogenics were represented as the percentage of maximal reflex in each mouse as the flexion forces differ from mouse to mouse. The control response was taken as the average of the three consecutive vehicle-induced responses in the beginning of each experiment. 2.4. Algogenic-induced biting and licking (ABL) test The algogenic-induced biting and licking (ABL) test was performed as described previously [18]. In the ABL test, the nociceptive biting and licking responses induced
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by i.pl. injection of different algogenic substances were measured. Mice were placed in a Plexiglas cage for 1 h to adapt to the environment. Before the test, mice were restrained in hand and gently taken inside a hard paper tube of internal diameter 2.5 cm. The right hind-paw was taken out of the tube and algogenic substances (BK, SP, ATP or ONO-54918-07) were injected into the paw in a volume of 20 l using a 30-gauge needle fitted to a Hamilton microsyringe. Mice were immediately put back to the cage and the time spent on biting and licking of the injected paw was measured with stopwatch for a period of 10 min after injection. Saline was injected as control. Mice were used only once. 2.5. Different conventional nociception tests In Hargreaves thermal paw withdrawal test, paw withdrawal latency was measured by exposing the right hind-paw to a thermal stimulus [4]. Unanaesthetized animals were placed in Plexiglas cages on top of a glass sheet and an adaptation period of 1 h was allowed. The thermal stimulus was positioned under the glass sheet to focus the projection bulb exactly on the middle of plantar surface of the animals and the latency to withdrawal of the paw was measured. Test was performed for 30 min at 10-min interval with a thermal latency of ∼10 s in na¨ıve mice. Paw pressure test was performed as described previously [1,13]. Mice were placed into a Plexiglas chamber on a 6 mm × 6 mm wire mesh grid floor and were allowed to accommodate for a period of 1 h. The mechanical stimulus was then delivered onto the middle of the plantar surface of the right hind-paw using a Transducer Indicator, and the withdrawal threshold was measured. Paw pressure test was performed for 30 min at 10-min interval with a pressure threshold of ∼10 g in na¨ıve mice. In the tail-flick test, animals were gently restrained by hand, and radiant heat was focused onto the marked black ventral surface of the tail as described previously [21]. The tail-flick response was evaluated for 30 min at 10-min interval with a thermal latency of ∼10 s in na¨ıve mice. In the formalin test, a 20-l aliquot of 1% formalin solution was administered into the hind-paw of na¨ıve mice with a Hamilton microsyringe, and the induced behaviors such as licking and biting of the injected paw were evaluated for 30 min as nociceptive responses [23]. 2.6. Measurement of plasma corticosterone level The plasma corticosterone level was measured as described previously with little modifications [7]. All nociception tests including the ANF test were performed for a period of 30 min. The mice were decapitated immediately after each nociception test and the whole blood was collected. The time interval between the noxious stimulus and manipulations until sacrifice were strictly maintained similar (1 min) among the different nociception tests. The
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plasma was separated by centrifuging at 3000×g for 15 min at 4 ◦ C. It was then collected into ice-chilled tubes containing 0.1% EDTA and stored at −80 ◦ C until assayed. The blood sample was collected between 10:00 and 11:00 h in order to exclude the effect of a circadian rhythm on circulating plasma corticosterone. The plasma coticosterone level was estimated fluorometrically according to the method of Zenker and Bernstein [24]. 2.7. Statistical analysis Results in the ANF test were analyzed with one-way analysis of variance and Dunnett’s post hoc test. Results in the ABL test were analyzed with one-way analysis of variance and Sheffe’s post hoc test. Statistical analysis of the plasma corticosterone data was made using the Student’s t-test. All data were presented as mean ± S.E.M. P values less than 0.05 were considered to indicate statistical significance.
3. Results 3.1. The ANF test is more sensitive to induce nociceptive response compared to the ABL test Fig. 1A and B shows the diagrammatic model of ANF test in mice and some representative traces of BK-induced nociceptive flexion responses, respectively. As shown in Fig. 1A, the mouse was taken into a soft cloth sling and three limbs were gently tied with soft cotton thread and the fourth limb (right hind) was connected to the lever of the isotonic transducer which was connected to a recorder. As shown in Fig. 1B, the mouse gave some biggest non-specific flexion responses immediately following cannulation producing some highest peaks on the recording sheet. After an adaptation period of about 5–10 min, i.pl. injection of vehicle saline did not produce any flexion response. However, i.pl. injection of 2 pmol of BK produced very sharp and stable nociceptive flexion responses injected at every 5-min interval without any test–retest effect (Fig. 1B). We next performed experiments to compare the doseresponse pattern of various algogenic substances in the ANF and ABL tests. In the ANF test, the initial flexion response induced within seconds upon i.pl. infusion of the algogenics was evaluated by normalizing it with the maximal flexion response in each mouse. The maximal flexion response was the biggest response among the non-specific flexion responses which occurred immediately following cannulation (Fig. 1B). We consider it as the maximum possible flexion capacity of the animal. The result has been represented as the percentage of the maximal flexion response as the flexion forces differ from mouse to mouse. The control vehicle response for each mouse was taken as the average responses upon three consecutive saline injections. As shown in Fig. 2A, i.pl. injection of BK produced dose-dependent
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Fig. 1. Model of the algogenic-induced nociceptive flexion (ANF) test in mice. (A) Diagrammatic model of the ANF test in mice. The mice were taken in soft cloth sling and three limbs were gently tied with soft cotton thread and the fourth limb (right hind) was connected to the lever of the isotonic transducer which was then connected to a recorder. The nociceptive flexion response induced by various algogenic substances was quantified as height in centimeters on a paper chart. (B) A representative trace of the bradykinin-induced flexion responses. After cannulation at right hind-paw, the mouse gave some of the biggest and non-specific flexion responses which diminished within minutes. After an adaptation period of about 10 min, i.pl. injection of saline did not produce any flexion response. On the other hand, repeated challenge of bradykinin (2 pmol, i.pl.) at every 5 min produced very sharp and stable flexion responses.
Fig. 2. Comparison between the algogenic-induced nociceptive flexion (ANF) and algogenic-induced biting and licking (ABL) tests in mice. (A) Dose-response curves for the bradykinin (BK)-induced type I nociceptive flexion responses in the ANF test and biting–licking responses in the ABL test. (B) Substance P (SP)-induced type I nociceptive flexion response in the ANF test and the biting–licking behavior in the ABL test. (C) Dose-dependent ATP-induced type II flexion response and biting–licking behavior in the ANF and ABL tests respectively. (D) Dose-response curves for the PGI2 agonist, ONO-54918-07-induced type III responses in the ANF test and biting–licking responses in the ABL test. In the ANF test, results are presented as percentage of maximal reflex (see Section 2 for details). In the ABL test, time spent (in seconds) in biting and licking of the injected paw for a period of 10 min after injection was measured. All data points represents mean ± S.E.M. The vertical bars indicate the standard error of the means. Number of animals for each data point in the ANF test was nine. The number of animals for each data point in the ABL test is indicated in parenthesis. For the ANF test, the data were analyzed using one-way ANOVA and Dunnett’s test for post hoc comparisons. For the ABL test, the data were analyzed using one-way ANOVA and Scheffe’s test for post hoc comparisons. ‘Veh’ means vehicle saline data. (∗ ) Indicates significant difference compared with the vehicle saline response at a P value of less than 0.05.
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nociceptive flexion responses from 0.1 fmol to 10 pmol through stimulation of neonatal capsaicin-sensitive type I fibers in the ANF test with an ED50 of 188.5 ± 53.2 fmol. BK produced significant flexion responses at doses of 100 fmol, 1 and 10 pmol compared with the control saline response (1.98 ± 0.95%) [F(6, 56) = 36.06; P < 0.0001]. In the ABL test, on the other hand, the total biting–licking time (in seconds) over a period of 10 min upon i.pl. injection of the algogenics was measured. No significant struggling or vocalization of the animal was observed after i.pl. injection of the algogenics in the ABL test. In this nociception test, i.pl. injection of BK produced dose-dependent nociceptive biting–licking response from 1 pmol to 10 nmol. However, significant biting–licking response was observed only with 10 nmol of BK compared with the vehicle saline (0.95 ± 0.32 s versus 25.40 ± 6.09 s) [F(3, 21) = 7.59; P = 0.001]. Similar to BK, i.pl. injection of SP, another stimulant of type I fibers, induced dose-dependent and potent nociceptive flexion responses in the ANF test from doses of 1 fmol to 10 pmol with an ED50 of 165.3 ± 18.7 fmol (Fig. 2B). SP produced significant flexion responses in the ANF test at doses of 10 and 100 fmol, 1 and 10 pmol compared with the control saline response (1.10 ± 0.56%) [F(5, 48) = 49.23; P < 0.0001]. On the other hand, in ABL test no significant biting–licking responses could be observed even with 10 nmol of SP compared with the vehicle saline (Fig. 2B) [F(2, 17) = 2.02; P = 0.16]. ATP, the stimulant of neonatal capsaicin-sensitive type II fibers also induced dose-dependent flexion responses from 1 fmol to 100 pmol in the ANF test with an ED50 of 36.9 ± 5.3 pmol (Fig. 2C). Significant flexion response compared with the vehicle saline response (1.22 ± 0.94%) was observed at 100 fmol, 1, 10 and 100 pmol [F(6, 56) = 58.73; P < 0.0001]. ATP also produced dose-dependent biting–licking response in the ABL test from doses of 100 pmol to 10 nmol (Fig. 2C). Significant biting–licking response was observed only at 10 nmol compared with vehicle saline injection (0.71 ± 0.34 s versus 23.48 ± 4.16 s) [F(3, 24) = 11.47; P < 0.0001]. The PGI2 agonist, ONO-54918-07 induced neonatal capsaicin-insensitive type III nociceptive responses from doses of 1 fmol to 100 pmol in the ANF test with the ED50 of 8.7 ± 2.7 pmol (Fig. 2D). ONO-54918-07 produced significant flexion responses in the ANF test at doses of 1, 10 and 100 pmol compared with the control saline response (1.44 ± 0.73%) [F(6, 56) = 23.19; P < 0.0001]. However, as shown in Fig. 2D, no significant biting–licking response could be observed with even 10 nmol of ONO-54918-07 in the ABL test compared with the vehicle saline (0.95±0.32 s versus 0.95 ± 0.58 s) [F(2, 13) = 0.15; P = 0.86]. Statistical comparisons of the responses in the ANF and ABL tests could not be performed due to their different natures. However, from the figures it is clearly evident that the initial active dose of all algogenics was much lower in the ANF test than in the ABL test, indicating better sensitivity of the ANF test to produce nociceptive response at extremely low stimulation.
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Fig. 3. Plasma corticosterone level after different nociception tests. The plasma corticosterone was measured in na¨ıve mice and in mice after different nociception tests. Immobilization of mice in a 50-ml conical tube for 30 min was used as positive control. In formalin test, 1% formalin (in 20 l) was injected into the right hind-paw. In tail-flick test, radiant heat was applied into ventral surface of the tail. In Hargreaves test, radiant heat was applied into the paw. In paw pressure test, pressure was applied into the paw. The ANF test was performed as described in Section 2. In control ANF (ANF-control) test, only saline was injected. In the ANF test with bradykinin (ANF-BK), 2 pmol of BK was injected i.pl. All tests were performed for 30 min. In all cases, the blood sample for corticosterone measurement was collected immediately following the test with no difference in time interval among the tests. Each data point represents mean ± S.E.M. from 12 mice. The vertical bars indicate the standard error of the means. Data were analyzed using Student’s t-test. (∗ ) Indicates significant difference compared with the control mice at P < 0.05.
3.2. Effects of various nociception tests on the plasma corticosterone level Circulating plasma corticosterone level is used as a measure of stress on animal. We measured the plasma corticosterone level in mice after the ANF test as well as some conventional nociception tests. As shown in Fig. 3, plasma corticosterone in control na¨ıve mice was 12.3 ± 1.6 g/dl. Immobilization of the animal in a 50-ml conical tube for 30 min served as positive control which caused an abrupt rise in plasma corticosterone level (40.1 ± 2.4 g/dl). When the plasma corticosterone level was measured after the formalin test, significant increase in corticosterone level in the mice was observed compared with the control mice (12.3 ± 1.6 g/dl versus 29.7 ± 1.6 g/dl; Student’s t-test, P < 0.05). The thermal tail-flick test also caused significant rise in plasma corticosterone level in mice compared with the control mice (12.3 ± 1.6 g/dl versus 28.6 ± 1.3 g/dl; Student’s t-test, P < 0.05). However, there was no significant increase in the corticosterone level in mice after the ANF test (ANF-BK: 16.91 ± 2.7 mg/dl), Hargreaves thermal nociception test (17 ± 2.9 mg/dl) or the mechanical
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paw pressure test (17.5 ± 1.9 g/dl) compared with the control na¨ıve mice (Student’s t-test, P > 0.05). There was no effect of the slight ether anesthesia in the ANF test on the plasma corticosterone level since similar results were obtained without this initial slight anesthesia (data not shown).
4. Discussion An ideal model of nociception should conform to certain characteristics including specificity, sensitivity, validity and reliability. We have already established the ANF test as a stimulus-specific and reliable method to study pain mechanism at periphery [5,19,20,22]. The method is also sufficiently sensitive to induce nociception by local application of extremely low doses of pain-producing substances. In the present report, we have reconfirmed the highly sensitive nature of this test. In most conventional chemical nociception tests after cutaneous application of algogenics, chemical stimulation occurs slowly after administration of algogenic substances. Thus, in such tests, the response is usually measured as behavioral scores in units of time. The ABL test falls under this category of chemical nociception test. On the other hand, the ANF test measures the flexion reflex response induced by different doses of an algogenic substance and the response occurs within the fraction of a second. When we compared the dose-response curves of different algogenic substances by these two types of nociception tests, it was evident that the ANF test is much more sensitive than the ABL test (Fig. 2A–D). The i.pl. application of BK into rodent’s paw is reported to produce both thermal and mechanical hyperalgesia at lower doses [17]. In our present report, i.pl. injection of BK induced a dose-dependent biting–licking behavior in the mice from 1 pmol to 10 nmol (Fig. 2A). However, significant biting–licking response was observed only at a dose of 10 nmol of BK compared with the vehicle saline injection (Fig. 2A). On the other hand, i.pl. injection of BK produced very sharp nociceptive flexion response from doses of 0.1 fmol to 10 pmol in the ANF test which is consistent with our previous reports [6,22]. Thus, BK was about 10,000-times more potent in the ANF test than in the ABL test. In other words, the ANF test was about 10,000-times more sensitive to produce BK-induced nociception than the ABL test. Similarly, SP, ATP and PGI2 agonist also induced dose-dependent nociceptive flexion reflex responses in the ANF test as reported previously [5,21]. The doses of SP, ATP or PGI2 agonist that produced flexion response were much lower than the doses needed to produce biting–licking responses (Fig. 2B–D). Although direct statistical comparison of the responses in the ANF and ABL tests could not be performed due to their different natures, we attempted to compare the two tests with regard to the initial active dose of different algogenics. As shown in Fig. 2A–D, the initial active dose of all algogenics was much lower in the ANF
test than in the ABL test, indicating better sensitivity of the ANF test to produce nociceptive response at extremely low stimulation. Thus, the initial significantly active dose of BK was 100 fmol in the ANF test while it was 10 nmol in the ABL test (Fig. 2A). This highly sensitive nature of the ANF test is reflected by the fact that, the nociceptive responses in this test appeared to involve relatively simple molecular and neuronal mechanisms, in contrast to the ABL test which use strong and sustained chemical stimuli leading to the release of several endogenous pain-producing substances, such as BK, SP, somatostatin, histamine and glutamate [11,12]. Experimental manipulations may radically alter the outcome of any behavioral model of nociception. Psychological stresses produce analgesic effects in both human and animals [2,3,10]. Such stress-induced analgesia has been reported to be either opioid- or nonopioid-mediated. On the other hand, anxiety has been regarded as an aggravating factor for clinical pain. Indeed, the facilitatory effect of anxiety on the action of morphine on a pain threshold was identified long ago [8]. Thus, it has become important to assess the effects of different manipulations on the experimental animals during the nociceptive tests. Increase in circulating plasma coricosterone level is used as an indication of acute stress on animals [15]. In this context, we have measured the circulating plasma corticosterone level in mice after the ANF test as well the other conventional tests of nociception. Interestingly, significant increase in plasma corticosterone level was observed after the formalin and tail-flick tests compared to plasma corticosterone level in control animals (Fig. 3). On the other hand, although little rise in plasma corticosterone was observed after the ANF, Hargreaves thermal nociception or mechanical paw pressure tests, they were statistically insignificant compared to the control level (Fig. 3). The plasma corticosterone level in the ANF test after saline and BK injection were not also significantly different (Fig. 3, last two columns). The significant rise in corticosterone level with the tail-flick test might be due to direct handling of animal during the test. On the other hand, the persistent pain on animal in formalin test may underlie the significant increase in corticosterone level in this test. However, relatively little stress on animals in the Hargreaves thermal test, mechanical paw pressure test or the ANF test is understood by the fact that there is no direct contact between the animals and the experimenter in these tests. In conclusion, we demonstrate that the ANF test is highly sensitive to detect pain signal at extremely low doses of algogenic substances. The simplicity of the neuronal network at the peripheral nerve ending makes the test more specific and sensitive than other conventional tests. Moreover, our results of plasma corticosterone level for the first time revealed the amount of stress on animals in different nociception tests. The ANF test has been found to produce almost no stress in animals due to experimental manipulations.
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