- Email: [email protected]
Increase of preprotachykinin mRNA in the uninjured mandibular neurons after rat infraorbital nerve transection
Neuroscience Letters 345 (2003) 57–60 www.elsevier.com/locate/neulet
Increase of preprotachykinin mRNA in the uninjured mandibular neurons after rat infraorbital nerve transection Kenzo Tsuzukia,b, Tetsuo Fukuokaa,*, Masafumi Sakagamib, Koichi Noguchia a
Department of Anatomy and Neuroscience, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan b Department of Otorhinolaryngology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan Received 26 February 2003; received in revised form 16 April 2003; accepted 19 April 2003
Abstract An increasing number of studies are suggesting that the adjacent uninjured primary afferents contribute to the mechanisms of neuropathic pain following peripheral nerve injury. In this study, we report that transection of the infraorbital nerve, a major branch of the maxillary nerve, causes exaggerated face grooming to normally innocuous mechanical stimuli in the skin territory of the uninjured mandibular nerve, and increases the expression of preprotachykinin mRNA in the primary afferent neurons in the mandibular zone in the trigeminal ganglia. Considering the various functions of substance P in the sensory transmission process, the increase in preprotachykinin mRNA in the uninjured primary afferent neurons may be one of the mechanisms of pain-related behavior in this neuropathic pain model. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Preprotachykinin; Activating transcription factor 3; Uninjured primary afferent neurons; Infraorbital nerve transection; Mechanical allodynia
Injury to peripheral nerves sometimes causes persistent pain. In order to study the etiology of this clinically important disorder, a series of experimental neuropathic pain models have been established [1,2,6,13]. These models involve partial injury of the sciatic nerve or its components that serve the hindlimb, therefore the primary afferent neurons in the dorsal root ganglion (DRG) are divided into directly injured neurons and uninjured ones. Recently, evidence has been accumulating for the idea that the uninjured DRG neurons have some role in the pathophysiology in these neuropathic pain models (for review, see Ref. [3]). On the other hand, the histochemical studies of the uninjured primary afferent neurons are still limited in number in trigeminal neuropathic pain models. Activating transcription factor 3 (ATF3) is a member of the activating transcription factor/cAMP-responsive element binding protein (ATF/CREB) family [5]. We have demonstrated that ATF3 is a useful marker to distinguish between injured and uninjured primary afferent neurons [14]. In a previous study, we have demonstrated that following infraorbital nerve (ION) transection, the uninjured trigeminal ganglion (TG) neurons increase the expression of * Corresponding author. Tel.: þ 81-798-45-6416; fax: þ81-798-45-6417. E-mail address: [email protected] (T. Fukuoka).
mRNA for the P2X3 receptor, one of the purine-gated cation channels [15]. In this study, we further examined the mechanical sensitivity of the skin territories of the injured ION and of the uninjured mandibular nerve (MANN), and quantified the expression of preprotachykinin (PPT) mRNA in the uninjured MANN region of the TG in this model. All experimental procedures conformed to the regulations of the Hyogo College of Medicine Committee on Animal Research and were carried out in accordance with the guidelines of NIH on animal care. A total of 27 male Sprague –Dawley rats (200 – 250 g) were used. A unilateral ION transection (n ¼ 11) or sham-operation (n ¼ 11) was performed as described in our previous report [15]. Six rats out of each group were used for behavioral testing, and five other rats of each group were used for the histochemical studies. Another five rats were used as naive controls in the histochemical study. For the behavioral test, two von Frey hairs were used: 4.36 mN (0.445 g) for the ION territory and 0.78 mN (0.080 g) for the MANN territory. These hairs were slowly applied to the appropriate territories on each side in the face until the hairs became slightly bent. For the behavioral scoring, we took the face grooming, a widely-used assessment of rat facial pain [16], induced by stimuli as an aversive response, and calculated the grooming frequency (%) induced over ten
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00487-7
58
K. Tsuzuki et al. / Neuroscience Letters 345 (2003) 57–60
trials (i.e. number of trials accompanied by face grooming/ 10 £ 100). To assess postoperative changes, repeated measures one-way ANOVA was performed, and the difference from the preoperative value was tested by Fisher’s protected least significant difference (PLSD). Two-tailed P values of , 0.05 were considered to be significant. Five rats from each group (ION transected, shamoperated, and naive groups) were processed for immunohistochemistry and in situ hybridization histochemistry (ISHH) 7 days after surgery. These animals did not receive behavioral testing in order to exclude the possibility that repeated mechanical stimuli might affect gene expression. They were deeply anesthetized with sodium pentobarbital and perfused transcardially with 1% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) (pH 7.4), followed by 4% PFA in 0.1 M PB. The bilateral trigeminal ganglia (TGs) were dissected out, post-fixed in the same fixative overnight for 12 h, and cryoprotected in 0.1 M PB containing 20% sucrose for another 12 h. The TGs were frozen, cut (14 mm thick), and thaw-mounted onto Vectabond- (Vector Lab. Inc., Burlingame, CA) coated slides. In order to distinguish the directly axotomized TG neurons from the uninjured ones, we examined ATF3 immunohistochemistry in the TG following ION transection using anti-ATF3 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as described in our previous report [15]. For the ISHH, an oligonucleotide probe complementary to bases 127 –171 [11] of the rat b-PPT-A mRNA was used [7]. The procedure for ISHH has been described in detail in our previous report [15]. From each rat, five to seven sections of the TGs were randomly selected. Measurements of the density of silver grains were performed using a computerized image analysis system (NIH image ver. 1.61) as described in our previous report [4]. Neurons with a grain density of ten-fold that of the background or greater were considered positively labeled. Comparisons among groups were made by one-way ANOVA followed by Fisher’s PLSD. Comparison between the ipsilateral and contralateral sides relative to the ION transection was tested by paired t-test. Two-tailed P values of , 0.05 were considered to be significant. Before surgery, application of a von Frey hair (0.445 g) to the skin territory of the ION almost always induced a face-grooming response. After unilateral ION transection, the ipsilateral territory clearly became hyposensitive to the stimulus (Fig. 1A). Although the contralateral sensitivity also slightly decreased, it was significantly higher than that of the ipsilateral side (P , 0:001 by repeated measure ANOVA). Before surgery, the grooming frequency caused by the application of a von Frey hair (0.08 g) to the MANN territory was 20– 30% (Fig. 1B). One day after unilateral ION transection, grooming frequency significantly increased not only on the ipsilateral side but also on the contralateral side. This hypersensitivity was transient and no significant increase in grooming frequency was seen
Fig. 1. The effect of ION transection on mechanical sensitivity of the face skin. Two von Frey filaments (bending force ¼ 0:445 and 0.08 g) were applied to the ION territory (A) and MANN territory (B), respectively. ION transection clearly caused mechanical hypoalgesia in the ipsilateral ION territory (closed circles in A), while minor changes were seen in the contralateral ION territory (open circles in A) and sham-operated ipsilateral ION territory (closed triangles in A). On the other hand, ION transection induced transient mechanical allodynia on the 1st day after surgery and delayed sustained mechanical allodynia after the 7th day following surgery in the spared ipsilateral MANN territory (closed circles in B). Only transient mechanical allodynia was seen in the contralateral ION territory on the 1st day after ION transection (open circles in B). No significant change was observed in the sham-operated ipsilateral ION territory (closed triangles in B). *P , 0:05; **P , 0:01; ***P , 0:001 vs. preoperative values (pre.) using repeated measure one-way ANOVA followed by Fisher’s PLSD.
thereafter on the contralateral side. In the territory of the MANN ipsilateral to ION transection, the delayed and sustained hypersensitivity was seen from the 7th day to at least the 28th day after surgery. No significant change was seen on the ipsilateral side of the sham-operated rats (Fig. 1B). Rat TG neurons are located in two regions in the ganglion: the MANN region (surrounded by an oval in Fig. 2A) and the remaining ophthalmic nerve and maxillary nerve (OPTN/MAXN) region. ATF3-immunoreactive nuclei were distributed exclusively in the OPTN/MAXN region (Fig. 2B), and no immunoreactive nucleus was seen in the MANN region in the TG ipsilateral to ION transection 7 days after surgery (Fig. 2C). These data certify that all neurons in the MANN region of the ipsilateral TG are spared from axotomy by ION transection. PPT mRNA was expressed by a subpopulation of the neurons throughout the TG. In the naive MANN region, 13.9 ^ 0.8% of the neurons expressed PPT mRNA. The percentage was significantly
K. Tsuzuki et al. / Neuroscience Letters 345 (2003) 57–60
59
Fig. 2. Immunohistochemistry for ATF3 of a TG 7 days after ION transection. The MANN region (the area surrounded by an oval) is located at the base of MANN and is easily distinguished from the remaining OPTN/MAXN region. ION is a major branch of the MAXN, therefore ATF3-immunoreactive nuclei were observed exclusively in the area of the OPTN/MAXN region (B), and no such nuclei are seen in the spared MANN region (C). Scale bar, 100 mm.
higher in the ipsilateral MANN region (Fig. 3A) as compared with the contralateral MANN region (Fig. 3B) in ION transected animals, and also with the ipsilateral MANN region in sham-operated animals (Fig. 3C). The PPT mRNA-expressing neurons were relatively small or medium in size (300 – 900 mm2) as compared to the total neurons (200 – 1500 mm2). On the other hand, ION transection induced profound reduction of PPT mRNA expression in the OPTN/MAXN region (data not shown). The present study demonstrated that ION transection induced mechanical allodynia of the skin territory innervated by the uninjured MANN and that the primary afferent neurons in the MANN region in the TGs increased the expression of b-PPT-A mRNA. Recently, Nomura et al. have reported a similar hypersensitivity of the uninjured nerve-innervating territory following inferior alveolar nerve transection in rats [12]. Taken with the hypoalgesia of the territory innervated by the directly injured nerve, the uninjured nerve components should be considered as one of the most attractive targets of study of abnormal pain after nerve injury. ION transection induced transient allodynia in the MANN territory on both sides at the 1st day after surgery and delayed and sustained allodynia on the ipsilateral side (Fig. 1B). The former may be caused by inflammation after surgery, because the MANN territory is near the skin incision. The latter allodynia was first seen at the 7th day after surgery when post-surgical inflammation had diminished. Therefore, the latter allodynia likely represents actual neuropathic pain. It is interesting that PPT mRNA expression increased at the same time point. b-PPT-A mRNA encodes two well-known neuropeptides: substance P (SP) and neurokinin A (NKA). There is a
Fig. 3. ISHH for PPT mRNA expression in the MANN region of the TGs 7 days after ION transection. More PPT mRNA-expressing neurons were seen on the ipsilateral side (A) as compared to the contralateral side (B). Scale bar, 100 mm. (C) Percentages of PPT mRNA-expressing neurons in the MANN region of the TGs of naive (n ¼ 5), ION transected (n ¼ 4), and sham-operated (n ¼ 5) rats. The percentage on the ipsilateral side (ipsi.) of ION transected rats was significantly higher than those on the same side of naive and sham-operated rats. *P , 0:05 by one-way ANOVA followed by Fisher’s PLSD. The percentage on the ipsilateral side of ION transected rats was significantly higher than that on the contralateral side (contra.). # P , 0:05 by paired t-test.
consensus that in the dorsal horn of the spinal cord, binding of primary afferent-derived SP to its receptor, the neurokinin (NK)-1 receptor, on the secondary spinal neurons is one of the most important mechanisms of central sensitization of spinal neurons and consequent exaggerated pain behavior [17]. These tachykinin mechanisms are less established in the trigeminal sensory system. However, there are NK-1 receptors in the superficial laminae of the caudal subnucleus of the spinal trigeminal nucleus (medullary dorsal horn) in which the central axons of the trigeminal primary afferents terminate [8]. Further, an NK-1 receptor antagonist reduces
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K. Tsuzuki et al. / Neuroscience Letters 345 (2003) 57–60
c-Fos expression within the trigeminal nucleus caudalis and thalamic neuronal activity, induced by C-fiber stimulation of the trigeminal nerve [10]. NKA and its receptor, the NK-2 receptor, have also been reported to have a role in some specific pain modalities in spinal dorsal horn [9], although there is no evidence that NKA modulates the function of the trigeminal nervous system. Taken together, we suggest that the uninjured primary afferent neurons play important roles in the trigeminal neuropathic pain model.
[8]
[9]
[10]
Acknowledgements [11]
This study was supported by Grants-in-Aid for Science Research from the Japanese Ministry of Education, Science and Culture.
[12]
References [1] G.J. Bennett, Y.-K. Xie, A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain 33 (1988) 87–107. [2] I. Decosterd, C.J. Woolf, Spared nerve injury: an animal model of persistent peripheral neuropathic pain, Pain 87 (2000) 149 –158. [3] T. Fukuoka, K. Noguchi, Contribution of the spared primary afferent neurons to the pathomechanisms of neuropathic pain, Mol. Neurobiol. 26 (2002) 57 –67. [4] T. Fukuoka, A. Tokunaga, E. Kondo, K. Miki, T. Tachibana, K. Noguchi, Change in mRNAs for neuropeptides and the GABA(A) receptor in dorsal root ganglion neurons in a rat experimental neuropathic pain model, Pain 78 (1998) 13 –26. [5] T. Hai, C.D. Wolfgang, D.K. Marsee, A.E. Allen, U. Sivaprasad, ATF3 and stress responses, Gene Expr. 7 (1999) 321–335. [6] S.H. Kim, J.M. Chung, An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat, Pain 50 (1992) 355– 363. [7] J.E. Krause, J.M. Chirgwin, M.S. Carter, Z.S. Xu, A.D. Hershey, Three rat preprotachykinin mRNAs encode the neuropeptides
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[15]
[16]
[17]
substance P and neurokinin A, Proc. Natl. Acad. Sci. USA 84 (1987) 881 –885. J.L. Li, D. Wang, T. Kaneko, R. Shigemoto, S. Nomura, N. Mizuno, The relationship between neurokinin-1 receptor and substance P in the medullary dorsal horn: a light and electron microscopic immunohistochemical study in the rat, Neurosci. Res. 36 (2000) 327 –334. X. Liu, J. Sandkuhler, Characterization of long-term potentiation of C-fiber-evoked potentials in spinal dorsal horn of adult rat: essential role of NK1 and NK2 receptors, J. Neurophysiol. 78 (1997) 1973–1982. J.C. Michaud, R. Alonso, C. Gueudet, M. Fournier, R. Calassi, J.C. Breliere, G. Le Fur, P. Soubrie, Effects of SR140333, a selective nonpeptide NK1 receptor antagonist, on trigemino-thalamic nociceptive pathways in the rat, Fundam. Clin. Pharmacol. 12 (1998) 88–94. K. Noguchi, M.A. Ruda, Gene regulation in an ascending nociceptive pathway: inflammation-induced increase in preprotachykinin mRNA in rat lamina I spinal projection neurons, J. Neurosci. 12 (1992) 2563–2572. H. Nomura, A. Ogawa, A. Tashiro, T. Morimoto, J.W. Hu, K. Iwata, Induction of Fos protein-like immunoreactivity in the trigeminal spinal nucleus caudalis and upper cervical cord following noxious and non-noxious mechanical stimulation of the whisker pad of the rat with an inferior alveolar nerve transection, Pain 95 (2002) 225 –238. Z. Seltzer, R. Dubner, Y. Shir, A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury, Pain 43 (1990) 205 –218. H. Tsujino, E. Kondo, T. Fukuoka, Y. Dai, A. Tokunaga, K. Miki, K. Yonenobu, T. Ochi, K. Noguchi, Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: a novel neuronal marker of nerve injury, Mol. Cell. Neurosci. 15 (2000) 170 –182. K. Tsuzuki, E. Kondo, T. Fukuoka, D. Yi, H. Tsujino, M. Sakagami, K. Noguchi, Differential regulation of P2X(3) mRNA expression by peripheral nerve injury in intact and injured neurons in the rat sensory ganglia, Pain 91 (2001) 351 –360. B.P. Vos, G. Hans, H. Adriaensen, Behavioral assessment of facial pain in rats: face grooming patterns after painful and non-painful sensory disturbances in the territory of the rat’s infraorbital nerve, Pain 76 (1998) 173 –178. C.J. Woolf, M.W. Salter, Neuronal plasticity: increasing the gain in pain, Science 288 (2000) 1765–1769.
Increase of preprotachykinin mRNA in the uninjured mandibular neurons after rat infraorbital nerve transection Kenzo Tsuzukia,b, Tetsuo Fukuokaa,*, Masafumi Sakagamib, Koichi Noguchia a
Department of Anatomy and Neuroscience, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan b Department of Otorhinolaryngology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan Received 26 February 2003; received in revised form 16 April 2003; accepted 19 April 2003
Abstract An increasing number of studies are suggesting that the adjacent uninjured primary afferents contribute to the mechanisms of neuropathic pain following peripheral nerve injury. In this study, we report that transection of the infraorbital nerve, a major branch of the maxillary nerve, causes exaggerated face grooming to normally innocuous mechanical stimuli in the skin territory of the uninjured mandibular nerve, and increases the expression of preprotachykinin mRNA in the primary afferent neurons in the mandibular zone in the trigeminal ganglia. Considering the various functions of substance P in the sensory transmission process, the increase in preprotachykinin mRNA in the uninjured primary afferent neurons may be one of the mechanisms of pain-related behavior in this neuropathic pain model. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Preprotachykinin; Activating transcription factor 3; Uninjured primary afferent neurons; Infraorbital nerve transection; Mechanical allodynia
Injury to peripheral nerves sometimes causes persistent pain. In order to study the etiology of this clinically important disorder, a series of experimental neuropathic pain models have been established [1,2,6,13]. These models involve partial injury of the sciatic nerve or its components that serve the hindlimb, therefore the primary afferent neurons in the dorsal root ganglion (DRG) are divided into directly injured neurons and uninjured ones. Recently, evidence has been accumulating for the idea that the uninjured DRG neurons have some role in the pathophysiology in these neuropathic pain models (for review, see Ref. [3]). On the other hand, the histochemical studies of the uninjured primary afferent neurons are still limited in number in trigeminal neuropathic pain models. Activating transcription factor 3 (ATF3) is a member of the activating transcription factor/cAMP-responsive element binding protein (ATF/CREB) family [5]. We have demonstrated that ATF3 is a useful marker to distinguish between injured and uninjured primary afferent neurons [14]. In a previous study, we have demonstrated that following infraorbital nerve (ION) transection, the uninjured trigeminal ganglion (TG) neurons increase the expression of * Corresponding author. Tel.: þ 81-798-45-6416; fax: þ81-798-45-6417. E-mail address: [email protected] (T. Fukuoka).
mRNA for the P2X3 receptor, one of the purine-gated cation channels [15]. In this study, we further examined the mechanical sensitivity of the skin territories of the injured ION and of the uninjured mandibular nerve (MANN), and quantified the expression of preprotachykinin (PPT) mRNA in the uninjured MANN region of the TG in this model. All experimental procedures conformed to the regulations of the Hyogo College of Medicine Committee on Animal Research and were carried out in accordance with the guidelines of NIH on animal care. A total of 27 male Sprague –Dawley rats (200 – 250 g) were used. A unilateral ION transection (n ¼ 11) or sham-operation (n ¼ 11) was performed as described in our previous report [15]. Six rats out of each group were used for behavioral testing, and five other rats of each group were used for the histochemical studies. Another five rats were used as naive controls in the histochemical study. For the behavioral test, two von Frey hairs were used: 4.36 mN (0.445 g) for the ION territory and 0.78 mN (0.080 g) for the MANN territory. These hairs were slowly applied to the appropriate territories on each side in the face until the hairs became slightly bent. For the behavioral scoring, we took the face grooming, a widely-used assessment of rat facial pain [16], induced by stimuli as an aversive response, and calculated the grooming frequency (%) induced over ten
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00487-7
58
K. Tsuzuki et al. / Neuroscience Letters 345 (2003) 57–60
trials (i.e. number of trials accompanied by face grooming/ 10 £ 100). To assess postoperative changes, repeated measures one-way ANOVA was performed, and the difference from the preoperative value was tested by Fisher’s protected least significant difference (PLSD). Two-tailed P values of , 0.05 were considered to be significant. Five rats from each group (ION transected, shamoperated, and naive groups) were processed for immunohistochemistry and in situ hybridization histochemistry (ISHH) 7 days after surgery. These animals did not receive behavioral testing in order to exclude the possibility that repeated mechanical stimuli might affect gene expression. They were deeply anesthetized with sodium pentobarbital and perfused transcardially with 1% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) (pH 7.4), followed by 4% PFA in 0.1 M PB. The bilateral trigeminal ganglia (TGs) were dissected out, post-fixed in the same fixative overnight for 12 h, and cryoprotected in 0.1 M PB containing 20% sucrose for another 12 h. The TGs were frozen, cut (14 mm thick), and thaw-mounted onto Vectabond- (Vector Lab. Inc., Burlingame, CA) coated slides. In order to distinguish the directly axotomized TG neurons from the uninjured ones, we examined ATF3 immunohistochemistry in the TG following ION transection using anti-ATF3 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as described in our previous report [15]. For the ISHH, an oligonucleotide probe complementary to bases 127 –171 [11] of the rat b-PPT-A mRNA was used [7]. The procedure for ISHH has been described in detail in our previous report [15]. From each rat, five to seven sections of the TGs were randomly selected. Measurements of the density of silver grains were performed using a computerized image analysis system (NIH image ver. 1.61) as described in our previous report [4]. Neurons with a grain density of ten-fold that of the background or greater were considered positively labeled. Comparisons among groups were made by one-way ANOVA followed by Fisher’s PLSD. Comparison between the ipsilateral and contralateral sides relative to the ION transection was tested by paired t-test. Two-tailed P values of , 0.05 were considered to be significant. Before surgery, application of a von Frey hair (0.445 g) to the skin territory of the ION almost always induced a face-grooming response. After unilateral ION transection, the ipsilateral territory clearly became hyposensitive to the stimulus (Fig. 1A). Although the contralateral sensitivity also slightly decreased, it was significantly higher than that of the ipsilateral side (P , 0:001 by repeated measure ANOVA). Before surgery, the grooming frequency caused by the application of a von Frey hair (0.08 g) to the MANN territory was 20– 30% (Fig. 1B). One day after unilateral ION transection, grooming frequency significantly increased not only on the ipsilateral side but also on the contralateral side. This hypersensitivity was transient and no significant increase in grooming frequency was seen
Fig. 1. The effect of ION transection on mechanical sensitivity of the face skin. Two von Frey filaments (bending force ¼ 0:445 and 0.08 g) were applied to the ION territory (A) and MANN territory (B), respectively. ION transection clearly caused mechanical hypoalgesia in the ipsilateral ION territory (closed circles in A), while minor changes were seen in the contralateral ION territory (open circles in A) and sham-operated ipsilateral ION territory (closed triangles in A). On the other hand, ION transection induced transient mechanical allodynia on the 1st day after surgery and delayed sustained mechanical allodynia after the 7th day following surgery in the spared ipsilateral MANN territory (closed circles in B). Only transient mechanical allodynia was seen in the contralateral ION territory on the 1st day after ION transection (open circles in B). No significant change was observed in the sham-operated ipsilateral ION territory (closed triangles in B). *P , 0:05; **P , 0:01; ***P , 0:001 vs. preoperative values (pre.) using repeated measure one-way ANOVA followed by Fisher’s PLSD.
thereafter on the contralateral side. In the territory of the MANN ipsilateral to ION transection, the delayed and sustained hypersensitivity was seen from the 7th day to at least the 28th day after surgery. No significant change was seen on the ipsilateral side of the sham-operated rats (Fig. 1B). Rat TG neurons are located in two regions in the ganglion: the MANN region (surrounded by an oval in Fig. 2A) and the remaining ophthalmic nerve and maxillary nerve (OPTN/MAXN) region. ATF3-immunoreactive nuclei were distributed exclusively in the OPTN/MAXN region (Fig. 2B), and no immunoreactive nucleus was seen in the MANN region in the TG ipsilateral to ION transection 7 days after surgery (Fig. 2C). These data certify that all neurons in the MANN region of the ipsilateral TG are spared from axotomy by ION transection. PPT mRNA was expressed by a subpopulation of the neurons throughout the TG. In the naive MANN region, 13.9 ^ 0.8% of the neurons expressed PPT mRNA. The percentage was significantly
K. Tsuzuki et al. / Neuroscience Letters 345 (2003) 57–60
59
Fig. 2. Immunohistochemistry for ATF3 of a TG 7 days after ION transection. The MANN region (the area surrounded by an oval) is located at the base of MANN and is easily distinguished from the remaining OPTN/MAXN region. ION is a major branch of the MAXN, therefore ATF3-immunoreactive nuclei were observed exclusively in the area of the OPTN/MAXN region (B), and no such nuclei are seen in the spared MANN region (C). Scale bar, 100 mm.
higher in the ipsilateral MANN region (Fig. 3A) as compared with the contralateral MANN region (Fig. 3B) in ION transected animals, and also with the ipsilateral MANN region in sham-operated animals (Fig. 3C). The PPT mRNA-expressing neurons were relatively small or medium in size (300 – 900 mm2) as compared to the total neurons (200 – 1500 mm2). On the other hand, ION transection induced profound reduction of PPT mRNA expression in the OPTN/MAXN region (data not shown). The present study demonstrated that ION transection induced mechanical allodynia of the skin territory innervated by the uninjured MANN and that the primary afferent neurons in the MANN region in the TGs increased the expression of b-PPT-A mRNA. Recently, Nomura et al. have reported a similar hypersensitivity of the uninjured nerve-innervating territory following inferior alveolar nerve transection in rats [12]. Taken with the hypoalgesia of the territory innervated by the directly injured nerve, the uninjured nerve components should be considered as one of the most attractive targets of study of abnormal pain after nerve injury. ION transection induced transient allodynia in the MANN territory on both sides at the 1st day after surgery and delayed and sustained allodynia on the ipsilateral side (Fig. 1B). The former may be caused by inflammation after surgery, because the MANN territory is near the skin incision. The latter allodynia was first seen at the 7th day after surgery when post-surgical inflammation had diminished. Therefore, the latter allodynia likely represents actual neuropathic pain. It is interesting that PPT mRNA expression increased at the same time point. b-PPT-A mRNA encodes two well-known neuropeptides: substance P (SP) and neurokinin A (NKA). There is a
Fig. 3. ISHH for PPT mRNA expression in the MANN region of the TGs 7 days after ION transection. More PPT mRNA-expressing neurons were seen on the ipsilateral side (A) as compared to the contralateral side (B). Scale bar, 100 mm. (C) Percentages of PPT mRNA-expressing neurons in the MANN region of the TGs of naive (n ¼ 5), ION transected (n ¼ 4), and sham-operated (n ¼ 5) rats. The percentage on the ipsilateral side (ipsi.) of ION transected rats was significantly higher than those on the same side of naive and sham-operated rats. *P , 0:05 by one-way ANOVA followed by Fisher’s PLSD. The percentage on the ipsilateral side of ION transected rats was significantly higher than that on the contralateral side (contra.). # P , 0:05 by paired t-test.
consensus that in the dorsal horn of the spinal cord, binding of primary afferent-derived SP to its receptor, the neurokinin (NK)-1 receptor, on the secondary spinal neurons is one of the most important mechanisms of central sensitization of spinal neurons and consequent exaggerated pain behavior [17]. These tachykinin mechanisms are less established in the trigeminal sensory system. However, there are NK-1 receptors in the superficial laminae of the caudal subnucleus of the spinal trigeminal nucleus (medullary dorsal horn) in which the central axons of the trigeminal primary afferents terminate [8]. Further, an NK-1 receptor antagonist reduces
60
K. Tsuzuki et al. / Neuroscience Letters 345 (2003) 57–60
c-Fos expression within the trigeminal nucleus caudalis and thalamic neuronal activity, induced by C-fiber stimulation of the trigeminal nerve [10]. NKA and its receptor, the NK-2 receptor, have also been reported to have a role in some specific pain modalities in spinal dorsal horn [9], although there is no evidence that NKA modulates the function of the trigeminal nervous system. Taken together, we suggest that the uninjured primary afferent neurons play important roles in the trigeminal neuropathic pain model.
[8]
[9]
[10]
Acknowledgements [11]
This study was supported by Grants-in-Aid for Science Research from the Japanese Ministry of Education, Science and Culture.
[12]
References [1] G.J. Bennett, Y.-K. Xie, A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain 33 (1988) 87–107. [2] I. Decosterd, C.J. Woolf, Spared nerve injury: an animal model of persistent peripheral neuropathic pain, Pain 87 (2000) 149 –158. [3] T. Fukuoka, K. Noguchi, Contribution of the spared primary afferent neurons to the pathomechanisms of neuropathic pain, Mol. Neurobiol. 26 (2002) 57 –67. [4] T. Fukuoka, A. Tokunaga, E. Kondo, K. Miki, T. Tachibana, K. Noguchi, Change in mRNAs for neuropeptides and the GABA(A) receptor in dorsal root ganglion neurons in a rat experimental neuropathic pain model, Pain 78 (1998) 13 –26. [5] T. Hai, C.D. Wolfgang, D.K. Marsee, A.E. Allen, U. Sivaprasad, ATF3 and stress responses, Gene Expr. 7 (1999) 321–335. [6] S.H. Kim, J.M. Chung, An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat, Pain 50 (1992) 355– 363. [7] J.E. Krause, J.M. Chirgwin, M.S. Carter, Z.S. Xu, A.D. Hershey, Three rat preprotachykinin mRNAs encode the neuropeptides
[13]
[14]
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