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Somatostatin inhibits activation of dorsal cutaneous primary afferents induced by antidromic stimulation of primary afferents from an adjacent thoracic segment in the rat
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
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Research Report
Somatostatin inhibits activation of dorsal cutaneous primary afferents induced by antidromic stimulation of primary afferents from an adjacent thoracic segment in the rat Yuan Guo a , Fan-Rong Yao a , Dong-Yuan Cao a,b , Joel G. Pickar b , Qi Zhang a , Hui-Sheng Wang a , Yan Zhao a,⁎ a
Department of Physiology and Pathophysiology, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061, P.R. China Palmer Center for Chiropractic Research, Palmer College of Chiropractic, 741 Brady Street, Davenport, IA 52803, USA
b
A R T I C LE I N FO
AB S T R A C T
Article history:
To investigate the effect of somatostatin on the cross-excitation between adjacent primary
Accepted 30 June 2008
afferent terminals in the rats, we recorded single unit activity from distal cut ends of dorsal
Available online 8 July 2008
cutaneous branches of the T10 and T12 spinal nerves in response to antidromic stimulation of the distal cut end of the T11 dorsal root in the presence and absence of somatostatin and
Keywords:
its receptor antagonist applied to the receptive field of the recorded nerve. Afferent fibers
Peripheral nerve terminal
were classified based upon their conduction velocity. Mean mechanical thresholds
Cross-excitation
decreased and spontaneous discharge rates increased significantly in C and Aδ but not Aβ
Hyperalgesia
fibers of the T10 and T12 spinal nerves in both male and female rats following antidromic
Somatostatin receptor
electrical stimulation (ADES) of the dorsal root from adjacent spinal segment (DRASS) indicating cross-excitation of thin fiber afferents. The cross-excitation was not significantly different between male and female rats. Microinjection of somatostatin into the receptive field of recorded units inhibited the cross-excitation. This inhibitory effect, in turn, was reversed by the somatostation receptor antagonist cyclo-somatostatin (c-SOM). Application of c-SOM alone followed by ADES of DRASS significantly decreased the mechanical thresholds and increased the discharge rates of C and Aδ fibers, indicating that endogenous release of somatostatin plays a tonic inhibitory role on the cross-excitation between peripheral nerves. These results suggest that somatostatin could inhibit the crossexcitation involved in peripheral hyperalgesia and have a peripheral analgesic effect. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
In the late nineteenth century, it was first realized that primary afferent fibers may serve an efferent role (reviewed
in Lynn, 1996). Experimental interventions show that primary afferents, in response to antidromic invasion of their receptive endings, can release neurotransmitters and neuropeptides into peripheral tissues causing vasodilatation, plasma protein
⁎ Corresponding author. Fax: +86 29 82656364. E-mail address: [email protected] (Y. Zhao). Abbreviations: ADES, antidromic electrical stimulation; c-SOM, cyclo-somatostatin; DRASS, dorsal root from adjacent spinal segment; SP, substance P; SST, somatostatin 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.06.111
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extravasation, and alter the spontaneous activity of neighboring peripheral nerve terminals (Lembeck, 1983; Holzer, 1988; Sluka et al., 1995; Millan, 1999; Willis, 1999; Carlton et al., 2001a, 2003). Modulation of primary afferent fibers is regarded as an important therapeutic target because sensitization of peripheral nociceptors defines the quality and duration of primary hyperalgesia during inflammation (Ji et al., 2006). To investigate the possibility that cross-excitation between peripheral afferent terminals could contribute to a peripheral mechanism of secondary hyperalgesia, we previously developed a model to observe potential changes in mechanical and electrophysiological receptive properties of adjacent afferent terminals following antidromic electrical stimulation (ADES) of a cut peripheral cutaneous nerve. Using this model, we demonstrated the existence of cross-excitation between adjacent primary afferent terminals via the non-synaptic release of some excitatory neurotransmitters (Zhao et al., 1996; Zhang et al., 2001, 2008; Jia et al., 2002; Sun et al., 2002, 2003; Cao et al., 2007). The findings are consistent with reports that secondary hyperalgesia is induced by antidromic stimulation of cutaneous nerves in man and rabbit (Lewis, 1936; Fitzgerald, 1979). Lewis (1936) proposed that the peripheral mechanism for secondary hyperalgesia was the sensitization of uninjured nociceptors arising from the peripheral release and spread of pro-inflammatory mediators from stimulated nociceptors. Both excitatory and inhibitory neurotransmitters can be released from primary afferent terminals under inflammatory and nociceptive conditions (Juranek and Lembeck, 1997; Elhassan et al., 2001; Tashev et al., 2001; Tsai et al., 2002; Du et al., 2003; Olias et al., 2004; Tian et al., 2005; Zhang et al., 2006a, 2006b; Cao et al., 2007; Zhang et al., 2008). Our previous studies indicate that excitatory neurotransmitters such as glutamate and substance P (SP) are involved in the crossexcitation between primary afferent terminals. Somatostatin (SST) and its synthetic octapeptide analog octreotide have been shown to have analgesic effects through activation of SST receptors (Silveri et al., 1994; Heppelmann and Pawlak, 1997; Elhassan et al., 2001; Tashev et al., 2001; Tsai et al., 2002; Olias et al., 2004; Paran et al., 2005; Sándor et al., 2006). In addition, SST and its receptors are found in presumed nociceptive neurons of the dorsal root ganglia (Lawson, 1995) and on peripheral unmyelinated nociceptive fibers (Elhassan et al., 1998; Patel, 1999; Carlton et al., 2001a, 2003). SST can be
released from capsaicin-sensitive sensory nerve terminals (Helyes et al., 2000, 2004; Pintér et al., 2006). These studies contribute to the idea that SST and its receptors have peripheral analgesic effects. The primary purpose of the present study was to investigate whether SST modulates the efferent activity of primary afferent neurons. Because some pain models report sex differences (Berkley et al., 2006; Greenspan et al. 2007), another purpose of this study was to determine if sex differences affect the SST modulation of efferent activity.
2.
Results
2.1.
Receptive characteristics of primary afferent fibers
A total of 172 single unit recordings were obtained. Of these units, 52 were C fibers, 84 were Aδ fibers, and 36 were Aβ fibers. Mean conduction velocities (CVs) used for classification, mean mechanical receptive thresholds, and spontaneous background discharge rates are presented in Table 1. The receptive fields of all units responded to mechanical stimulation and were located 0.5 to 2.0 cm from the dorsal midline. All units exhibited very low spontaneous background discharge rates and there was no significant difference in spontaneous discharge rates among the 3 types of units (P N 0.05, one way ANOVA test followed by Dunnett's method) and there was no significant difference between male and female rats (P N 0.05, t-test).
2.2. Cross-excitation after ADES of the dorsal root from adjacent spinal segment (DRASS) In the group receiving only ADES of the DRASS (St), after stimulating the T11 dorsal root, mean mechanical thresholds of C units from the T10 and T12 spinal nerve in male and female rats decreased significantly from 0.80 ± 0.10 and 0.63 ± 0.07 mN to 0.58 ± 0.04 and 0.50 ± 0.04 mN, respectively (P b 0.01, paired t-test, shown in Fig. 1A). Mean discharge rates of C units in male and female rats significantly increased from 1.35 ± 0.15 and 1.50 ± 0.21 impulse/min to 3.08 ± 0.48 and 3.17 ± 0.35 impulse/min, respectively compared with control before the stimulation (P b 0.05, paired t-test, shown in Fig. 1B). The increased discharge rates lasted for at least 10 min following
Table 1 – Baseline characteristics of primary afferent fibers C units
Mean mechanical receptive threshold (mN) Mean conduction velocity (m/s) Spontaneous background discharge rate (impulse/min)
Aδ units
Aβ units
Male rats (n = 20)
Female rats (n = 32)
Male rats (n = 37)
Female rats (n = 47)
Male rats (n = 14)
Female rats (n = 22)
0.76 ± 0.11
0.62 ± 0.08
0.29 ± 0.05
0.26 ± 0.02
0.22 ± 0.02
0.21 ± 0.02
1.45 ± 0.07 (0.86–1.84) 1.35 ± 0.15
1.20 ± 0.07 (0.75–1.72) 1.50 ± 0.21
10.29 ± 1.77 (2.14–28.30) 1.04 ± 0.17
40.56 ± 1.83 (32.50–48.60) 1.04 ± 0.26
40.85 ± 2.41 (31.10–54.30) 0.64 ± 0.10
9.97 ± 1.00 (2.56–26.70) 0.83 ± 0.17
All values given as mean ± SEM. Values in parentheses show the range of conduction velocity.
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trical stimulation of the T11 dorsal root in male or in female rats (P N 0.05, paired t-test, Fig. 1 and 2C).
2.3.
Fig. 1 – Changes in mean mechanical threshold (A) and discharge rates (B) of C, Aδ and Aβ units following the antidromic electrical stimulation of dorsal root from adjacent spinal segment in male and female rats. M: in male rats; F: in female rats. *P < 0.05, compared with those before stimulation. (n = 20 for C units in male and n = 32 for C units in female rats, n = 37 for Aδ units in male rats, n = 47 for Aδ units in female rats, n = 14 for Aβ units in male rats and n = 22 for Aβ units in female rats).
ADES, gradually returning to the pre-stimulation level thereafter. No significant difference in spontaneous discharge rate was found between males and females (P N 0.05, t-test). Fig. 2A is an original recording showing the discharges of an activated C unit after ADES of DRASS. For Aδ units in male and female rats, the mean mechanical thresholds decreased significantly from 0.30 ± 0.03 and 0.25 ± 0.02 mN to 0.25 ± 0.02 and 0.21 ± 0.01 mN, respectively (P b 0.05, paired t-test, shown in Fig. 1A). Similar to C units, mean discharge rates significantly increased from 1.04 ± 0.17 and 0.83 ± 0.17 impulse/min to 2.51 ± 0.36 and 2.21 ± 0.30 impulse/ min after ADES (P b 0.05, paired t-test, Fig. 1B), and no significant difference was found between males and females (P N 0.05, t-test). Fig. 2B shows the discharges of an activated Aδ unit following antidromic stimulation. Mean mechanical thresholds and discharge rates of Aβ units showed no obvious change following antidromic elec-
Effects of SST on cross-excitation
In the group receiving normal saline injection followed by ADES of DRASS (Ns + St), mean mechanical thresholds of C units in male and female rats decreased from 0.68 ± 0.13 and 0.66 ± 0.09 mN to 0.51 ± 0.07 and 0.46 ± 0.05 mN (P b 0.01, paired t-test, Fig. 3A and B), similar to that in the St group. Following ADES, mean discharge rates of C units in male rats significantly increased from 1.20 ± 0.25 to 3.06 ± 0.75 impulse/min and in female rats from 0.83 ± 0.14 to 2.35 ± 0.28 impulse/min (P b 0.05, paired t-test, Fig. 4A and B), again similar to the St group. In the group receiving an injection of SST followed by ADES of DRASS (Sst + St), mean mechanical thresholds of C units in male and female rats were 0.66 ± 0.13 and 0.64 ± 0.09 mN, which were not different from the thresholds before stimulation (0.71 ± 0.07 and 0.62 ± 0.07 mN, P N 0.05, paired t-test, Fig. 3A and B). Unlike the St group, mean discharge rates following ADES were 1.84 ± 0.30 impulse/ min for C units in male rats and 1.23 ± 0.16 impulse/min for C units in female rats, not significantly different from the control discharge rates measured before ADES (1.20 ± 0.31 and 0.79 ± 0.13 impulse/min, P N 0.05, paired t-test, shown in Fig. 4A and B). In the Ns + St group following ADES, mean discharge rates of male and female Aδ units significantly increased from 0.95 ± 0.25 and 0.96 ± 0.12 impulse/min to 2.46 ± 0.63 and 2.20 ± 0.23 impulse/min, respectively (P b 0.05, paired t-test, Fig. 4C and D). In the Sst + St group, mean discharge rates of Aδ units following ADES were 1.37 ± 0.26 and 1.11 ± 0.15 impulse/min for male and female rats, respectively. No significant difference was found when compared with that before ADES (1.00 ± 0.20 and 0.65 ± 0.09, P N 0.05, paired t-test, Fig. 4C and D). After antidromic stimulation, mean mechanical thresholds of Aδ units in male and female rats significantly decreased from 0.28 ± 0.05 and 0.26 ± 0.02 mN to 0.24 ± 0.03 and 0.21 ± 0.02 mN in the Ns +St group (P b 0.05, paired t-test, Fig. 3C and D), but not in the Sst + St group (P N 0.05, paired t-test, shown in Fig. 3C and D). These results suggest that local administration of SST inhibited the cross-excitation of C and Aδ units evoked by ADES of DRASS. The inhibitory effect of SST on afferent discharge rates was not significantly different between male and female rats for C and Aδ fibers (P N 0.05, t-test). For Aβ units in the Ns + St group, mean mechanical thresholds and discharge rates did not change significantly following ADES. SST had no effect on either the mean mechanical thresholds or on the mean discharge rates of Aβ units following ADES (P N 0.05, paired t-test, Fig. 3E, F, 4E and F).
2.4. Effects of an SST receptor antagonist on cross-excitation In the group receiving co-injection of the SST receptor antagonist cyclo-somatostatin (c-SOM) prior to SST injection then followed by ADES of DRASS (c-som + Sst + St), antidromic stimulation decreased mean mechanical thresholds of
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Fig. 2 – Original recordings of discharge from a C unit (A), an Aδ unit (B) and an Aβ unit (C) following antidromic electrical stimulation of dorsal root from adjacent spinal segment. Conduction velocities of the units were 0.77, 17.7 and 43.7 m/s for C, Aδ and Aβ unit, respectively.
male and female C units from 0.60 ± 0.09 and 0.59 ± 0.12 mN to 0.41 ± 0.06 and 0.48 ± 0.12 mN (P b 0.01, pair t-test, Fig. 3A and B), and significantly increased mean discharge rates from 0.83 ± 0.38 and 1.13 ± 0.29 impulse/min to 3.83 ± 0.67 and
2.20 ± 0.61 impulse/min following ADES (P b 0.05, pair t-test, Fig. 4A and B). Mean mechanical thresholds of Aδ units in male and female rats decreased from 0.30 ± 0.06 and 0.28 ± 0.03 mN to
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Fig. 3 – Changes in mechanical threshold after antidromic electrical stimulation of the dorsal root from adjacent spinal segment in all groups. (A) C units in male rats; (B) C units in female rats; (C) Aδ units in male rats; (D) Aδ units in female rats; (E) Aβ units in male rats; (F) Aβ units in female rats.*P < 0.05, **P < 0.01, compared with those before stimulation.
0.22 ± 0.04 and 0.23 ± 0.03 mN after ADES, respectively (P b 0.01, pair t-test, Fig. 3C and D). Mean discharge rates significantly increased from 0.71 ± 0.31 and 1.13 ± 0.24 impulse/min to 3.69 ± 0.97 and 3.76 ± 0.66 impulse/min, respectively (P b 0.05, paired t-test, Fig. 4C and D). These data indicate that the SST receptor antagonist reversed the inhibitory effects of SST. In the c-som + Sst + St group, neither the mean mechanical thresholds nor the mean discharge rates of Aβ units significantly changed following ADES in male and female rats (P N 0.05, paired t-test, Fig. 3E, F, 4E and F).
2.5.
Tonic inhibitory effect of SST on the cross-excitation
In the group receiving an injection of c-SOM followed by ADES of DRASS (c-som + St), mean mechanical thresholds of C units in male and female rats significantly decreased from 0.64 ± 0.06 and 0.57 ± 0.12 mN to 0.44 ± 0.07 and 0.46 ± 0.13 mN, and mean mechanical thresholds of Aδ units significantly decreased from 0.29 ± 0.04 and 0.26 ± 0.04 mN to 0.22 ± 0.03 and 0.20 ± 0.03 mN in male and female rats following ADES (P b 0.01, paired t-test, Fig. 3A–D). Mean discharge rates in C units significantly increased from 1.50 ± 0.49 and 0.97 ± 0.22
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Fig. 4 – Changes in discharge rate after antidromic electrical stimulation of dorsal root from adjacent spinal segment in all groups. (A) C units in male rats; (B) C units in female rats; (C) Aδ units in male rats; (D) Aδ units in female rats; (E) Aβ units in male rats; (F) Aβ units in female rats. *P < 0.05, compared with those before stimulation.
impulse/min to 5.71 ± 1.60 and 4.41 ± 0.85 impulse/min in male and female rats, and mean discharge rates in Aδ units significantly increased from 1.17 ± 0.48 and 0.91 ± 0.20 impulse/min to 3.87 ± 1.14 and 4.40 ± 0.88 impulse/min in male and female rats following stimulation (P b 0.01, paired t-test, Fig. 4A–D). The mean changes in discharge rates (percentage of net change from control) for C and Aδ units
following ADES in the c-som + St group were significantly greater than that in the St and Ns + St groups (P b 0.05, one way ANOVA test followed by Dunnett's method, Fig. 5A). The mean changes in mechanical thresholds (percentage of net change from control) for C and Aδ units following ADES in the c-som + St group were significantly different from that in the St and Ns + St groups (P b 0.05, one way ANOVA test followed
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by Dunnett's method, Fig. 5B). The results suggested that SST might have been released tonically in peripheral tissues and exerted inhibitory effects on peripheral afferent terminals. For Aβ units, SST had no effect on either the mean mechanical thresholds or on the mean discharge rates in male and female rats following ADES in the c-som + St group (P N 0.05, paired t-test, Fig. 3E, F, 4E and F).
3.
Discussion
The most important finding from the present study is that local injection of SST into the receptive field of the recorded afferent nerve blocked the increase in discharge rate and the decrease in mechanical threshold of C and Aδ fibers that resulted from ADES of DRASS. In our previous studies, we demonstrated that primary afferent terminals could be activated by antidromic stimulation of an adjacent peripheral cutaneous nerve (Zhao et al., 1996; Zhang et al., 2001, 2008; Jia et al., 2002; Sun et al., 2002, 2003; Cao et al., 2007). The experimental model used in the present study had character-
Fig. 5 – The differences in mean changes in discharge rates and mechanical thresholds (percentage of net change from control) between the Ns + St and the c-som + St groups, and between the St and the c-som + St groups. (A) The mean changes of discharge rates in three types of fibers; (B) The mean changes percentage in mechanical thresholds in three types of fibers. *, P < 0.05.
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istics similar to that used in our previous studies for studying cross-excitation between primary afferent terminals in vivo. The only difference is that in the present study the dorsal root was stimulated, eliminating the possibility that cross-excitation was caused by activation of sympathetic efferents since many studies have reported the existence of sympatheticsomatosensory interactions or coupling in the peripheral nervous system (Chung et al., 1997; Baron, 2000; Xie et al., 2007). As described in previous studies (Lembeck, 1983; Holzer, 1988; Du et al., 2003), when a primary afferent fiber is excited by inflammation, injury or antidromic stimulation, some neurotransmitters and neuromodulators can be released from terminals in branches of the same fiber by the axon reflex. It seems conceivable that some neurotransmitters could also diffuse and bind to receptors on terminals of neighboring afferent fibers. Our previous studies indicate that the mean discharge rate of primary afferents significantly increases after antidromic electrical stimulation of an adjacent spinal nerve and that local injection of NMDA, nonNMDA or NK-1 receptor antagonists into the receptive field of the recorded units blocks the increase of discharge rate (Cao et al., 2007; Zhang et al., 2008). Antidromically-activated release of neurotransmitters/neuromodulators may be a neural basis for the peripheral sensitization of pain, especially for secondary hyperalgesia. It has been widely accepted that some neurotransmitters/ neuromodulators are released from unmyelinated and thinly myelinated nerve fibers following antidromic stimulation. These neurotransmitters/neuromodulators include proinflammatory mediators such as glutamate (Juranek and Lembeck, 1997; Omote et al., 1998; deGroot et al., 2000), SP, calcitonin gene-related peptide (Lembeck, 1983; Holzer, 1988; Sakaguchi et al., 1991; Juranek and Lembeck, 1997; McGillis and Fernandez, 1999), as well as anti-inflammatory mediators such as opioids (Cabot et al., 1997; Bigliardi-Qi et al., 2004) and SST (Szolcsányi et al., 1998; Carlton et al., 2001a, 2003; Helyes et al., 2004). The balance between releases of these pro- and antiinflammatory mediators may contribute to the resolution of local inflammation and hyperalgesia in peripheral tissues. Intraplantar injection of SST or the SST analogue octreotide reduces both nociceptive behaviors induced by carrageenan and formalin and responses to thermal stimulation of Cmechanoheat sensitive fibers in a dose-dependent fashion (Corsi et al., 1997; Carlton et al., 2001a, 2001b, 2003, 2004). SST also reduces responses of joint mechanoreceptors to noxious rotation of the cat knee joint and to both innocuous and noxious rotation of the inflamed knee joint (Heppelmann and Pawlak, 1997; Pawlak and Schmidt, 2004). In the present study, the effects of SST were blocked by the SST receptor antagonist c-SOM, suggesting receptor specificity in the process. In addition, ADES in the presence of c-SOM produced a greater increase in discharge rate and a greater decrease in mechanical threshold compared with ADES and saline vehicle strongly suggesting that SST was tonically released producing a cross-excitatory inhibition at peripheral nerve terminals. The results are consistent with previous studies demonstrating that SST has analgesic effects in rodents and humans by acting on peripheral SST receptors (Karalis et al., 1994; Silveri et al., 1994; Helyes
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et al., 2000, 2004; Corsi et al., 1997; Olias et al. 2004; Szolcsányi et al., 2004; Paran et al., 2005; Ji et al., 2006). SST receptors have been identified on afferent terminals and in the dorsal root ganglion (Elhassan et al., 1998; Patel, 1999; Carlton et al., 2001a, 2003). A deficiency in the sst2 subtype of SST receptor is associated with exaggerated jejunal afferent sensitivity to both mechanical and chemical stimulation in mice (Rong et al., 2007). SST, after peripheral release from nerve terminals, may affect not only other peripherally released neuropeptides but cells from other physiological systems as well. SST and the pro-inflammatory neuropeptide SP co-exist in the same neuron (Kashiba et al., 1996) and separate in different populations of primary afferent neurons (Hökfelt et al., 1976). Under inflammatory conditions, SST may inhibit the release of pro-inflammatory neuropeptides from sensory terminals and thereby attenuate nociceptive responses (Green et al., 1992; Hathway et al., 2001; Carlton et al., 2001a, 2001b; Sawynok, 2003; Weckbecker et al., 2003; Grilli et al., 2004). SST also acts on receptors of vascular endothelial and immune cells and may form communication networks between the nervous and immune systems affecting both inflammation and nociception (Krantic, 2000; Dasgupta, 2004; Pintér et al., 2006). Some studies indicate females have lower thresholds to noxious stimuli, a greater ability to discriminate stimuli, higher pain ratings, and less tolerance for noxious stimuli compared to males (Berkley et al., 2006; Greenspan et al. 2007). Injection of glutamate into the masseter muscle induces a muscle pain response which is significantly greater in females than in males (Cairns et al., 2001, 2003). Subcutaneous administration of glutamate in human subjects evokes sex-related differences in some pain responses (Gazerani et al., 2006). On the other hand, a number of studies indicate the absence of sex differences in some pain responses (Hogeweg et al., 1996; Banik et al., 2006). In the present study no obvious sex differences were present in the cross-excitation of peripheral afferent terminals following ADES of the dorsal root from the adjacent segment. In summary, the present study suggests that crossexcitation between peripheral afferent terminals exists in both male and female rats and that somatostatin has a receptor-mediated tonic inhibitory effect on the crossexcitation. Local analgesic effects of somatostatin produced by local peripheral delivery may avoid deleterious side effects in the central nervous system and provide a basis for the use of somatostatin or its analogs in the pharmacotherapy of acute and chronic painful inflammatory diseases of peripheral origin.
4.
Experimental procedures
4.1.
Animals
Experiments were performed on 97 Sprague–Dawley rats weighing 220–300 g. The animals were housed on a 12–12 h light-dark cycle, and given food and water available ad libitum. Experimental procedures were approved by the
Institutional Animal Ethics Committee of Xi'an Jiaotong University.
4.2.
Surgical procedures
Rats were anesthetized initially with urethane (1.0 g/kg i.p.); supplemental doses (0.05 g/kg/h) were given as needed to maintain areflexia. Hair on the back was removed by shaving. In order to avoid damaging receptive fields on the back, a left paramedian skin incision 1 cm away from the median line was made longitudinally to expose the right dorsal cutaneous branches of the T10 and T12 spinal nerves. The cutaneous branches were dissected and freed for 2.5–3.5 cm from the surrounding tissues. A T10-12 laminectomy was performed in order to expose and cut the right T11 dorsal root for antidromic electrical stimulation (ADES) (see Fig. 6). A pool was formed by raising the skin flaps and filling the space with warm paraffin oil (37 °C). Rectal temperature was maintained at approximately 37 ± 0.5 °C using a servo-controlled heating blanket. The isolated T10 and T12 cutaneous nerve branches were cut close to their passage through the latissimus dorsi muscle in order to block the afferent and efferent information to and from the spinal cord.
4.3.
Stimulation and recording
The distal cut end of the T11 dorsal root was placed across silver bipolar electrodes for ADES. On a small platform, the distal cut portion of the dorsal cutaneous branches of the T10 or T12 spinal nerves were mechanically desheathed and teased apart under a dissecting microscope. Small filaments were repeatedly split with sharpened watchmaker forceps until single unit activity was isolated. Neural activity from T10 or T12 was recorded using platinum bipolar electrodes, then amplified (Biophysical Amplifier AVB-11A), filtered (30– 3000 Hz), and displayed on an oscilloscope (VC-11, Nihon Kohden, Japan) for monitoring the action potential's waveform and amplitude. The signals were also fed into a computer based data acquisition system (Spike2, Cambridge Electronic Design Limited, Cambridge, UK) that allowed continuous monitoring of discharges as well as off-line data analysis. Fig. 6 summarizes the experimental approach and demonstrates the cross-excitation induced by ADES of DRASS. For all groups discharge rates prior to any intervention were recorded for 2 min and served as control values. Then the T11 dorsal root was electrically stimulated continuously for 30 s (intensity 0.6 mA, duration 0.5 ms, frequency 20 Hz). In previous references and our preliminary experiments we found this intensity excited most Aδ and some C fibers (Leem et al., 1993; Zhao et al., 1996; Kanda et al., 2001) Following ADES, discharge rates were recorded for an additional 10 min. The mechanical threshold of each unit was measured with calibrated von Frey's filaments applied to its receptive field (RF) and expressed as the minimum force (mN) needed to evoke a response in ≥50% of the trials (Cain et al., 2001; Tian et al., 2005; Zhang et al., 2006a, 2006b). The location of each unit's receptive field that was most sensitive to mechanical stimulation was marked by a felt tip pen and
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vehicle (10 μl) was slowly injected over a 5-min period after recording background discharge.
4.5.
Fig. 6 – Schematic drawing showing the experimental method and the presumed pathway for cross-excitation between peripheral nerve terminals. When afferent nerves are excited by inflammation, injury or antidromic stimulation, excitatory and inhibitory neurotransmitters can be released from peripheral terminals of the afferent nerves. Neurotransmitters released from peripheral terminals may diffuse to adjacent afferent terminals and influence the activity of adjacent terminals.
was the target for drug injection. Mechanical thresholds were determined before and after ADES. The range of von Frey filaments used in this study exerted bending forces from 0.1 mN to 42.3 mN. Primary afferents were classified by their CV. A single electrical stimulus (square wave) with progressively increasing intensity (0.1–1 mA, duration 0.5 ms) was applied to the receptive field using silver bipolar electrodes. CV was calculated by dividing the distance from the stimulating to the recording electrode by the latency of the action potential. Units with CVs b2.0 m/s were classified as C fibers, 2.0–30.0 m/s as Aδ fibers, and 30.1–70.0 m/s as Aβ fibers (Horch et al., 1977; Tian et al., 2005; Zhang et al., 2006a, 2006b).
4.4.
Groups and pharmacological intervention
Five experimental groups were used: 1) ADES of DRASS only (St); 2) normal saline injection followed by ADES of DRASS (Ns + St); 3) SST injection followed by ADES of DRASS (Sst + St); 4) co-injection of SST receptor antagonist cyclo-somatostatin (c-SOM) prior to SST and followed by ADES of DRASS (c-som + Sst + St) and 5) injection of c-SOM followed by ADES of DRASS (c-som + St). SST (Sigma Co., St. Louis, MO, USA) was diluted with saline to 0.1 mM (Heppelmann and Pawlak, 1997). c-SOM (Sigma Co., St. Louis, MO, USA) was diluted with saline to 12.8 μM (according to preliminary experiments). In order to inject drugs or vehicle into the receptive field of a recorded unit, we attached a needle (28-gauge) to a 50 μl Hamilton syringe via PE10 tubing and inserted the needle subcutaneously into most sensitive region of the receptive field. Drug or the saline
Data analysis
Discharge rates before and after ADES were counted at one minute intervals and expressed in impulse/min. All data were presented as mean ± SEM. Statistical analysis was performed with SigmaStat 2.0 software. One way ANOVA test followed by Dunnett's method was used to compare differences in spontaneous background discharge rates among the 3 types of units and differences in discharge rates and mechanical thresholds among St, Ns + St and c-som + St groups. Paired ttests were used to compare mean mechanical thresholds before and after ADES, and to compare mean discharge rates before and after ADES. An t-test was used to compare mechanical thresholds and discharge rates between male and female rats. A P-value of b0.05 was considered statistically significant.
Acknowledgments The project was supported by the National Natural Science Foundation of China (No. 30600219, 30772705) and the Special Research Fund for the Doctoral Program of High Education (No. 20070698101). REFERENCES
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Somatostatin inhibits activation of dorsal cutaneous primary afferents induced by antidromic stimulation of primary afferents from an adjacent thoracic segment in the rat Yuan Guo a , Fan-Rong Yao a , Dong-Yuan Cao a,b , Joel G. Pickar b , Qi Zhang a , Hui-Sheng Wang a , Yan Zhao a,⁎ a
Department of Physiology and Pathophysiology, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi 710061, P.R. China Palmer Center for Chiropractic Research, Palmer College of Chiropractic, 741 Brady Street, Davenport, IA 52803, USA
b
A R T I C LE I N FO
AB S T R A C T
Article history:
To investigate the effect of somatostatin on the cross-excitation between adjacent primary
Accepted 30 June 2008
afferent terminals in the rats, we recorded single unit activity from distal cut ends of dorsal
Available online 8 July 2008
cutaneous branches of the T10 and T12 spinal nerves in response to antidromic stimulation of the distal cut end of the T11 dorsal root in the presence and absence of somatostatin and
Keywords:
its receptor antagonist applied to the receptive field of the recorded nerve. Afferent fibers
Peripheral nerve terminal
were classified based upon their conduction velocity. Mean mechanical thresholds
Cross-excitation
decreased and spontaneous discharge rates increased significantly in C and Aδ but not Aβ
Hyperalgesia
fibers of the T10 and T12 spinal nerves in both male and female rats following antidromic
Somatostatin receptor
electrical stimulation (ADES) of the dorsal root from adjacent spinal segment (DRASS) indicating cross-excitation of thin fiber afferents. The cross-excitation was not significantly different between male and female rats. Microinjection of somatostatin into the receptive field of recorded units inhibited the cross-excitation. This inhibitory effect, in turn, was reversed by the somatostation receptor antagonist cyclo-somatostatin (c-SOM). Application of c-SOM alone followed by ADES of DRASS significantly decreased the mechanical thresholds and increased the discharge rates of C and Aδ fibers, indicating that endogenous release of somatostatin plays a tonic inhibitory role on the cross-excitation between peripheral nerves. These results suggest that somatostatin could inhibit the crossexcitation involved in peripheral hyperalgesia and have a peripheral analgesic effect. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
In the late nineteenth century, it was first realized that primary afferent fibers may serve an efferent role (reviewed
in Lynn, 1996). Experimental interventions show that primary afferents, in response to antidromic invasion of their receptive endings, can release neurotransmitters and neuropeptides into peripheral tissues causing vasodilatation, plasma protein
⁎ Corresponding author. Fax: +86 29 82656364. E-mail address: [email protected] (Y. Zhao). Abbreviations: ADES, antidromic electrical stimulation; c-SOM, cyclo-somatostatin; DRASS, dorsal root from adjacent spinal segment; SP, substance P; SST, somatostatin 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.06.111
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extravasation, and alter the spontaneous activity of neighboring peripheral nerve terminals (Lembeck, 1983; Holzer, 1988; Sluka et al., 1995; Millan, 1999; Willis, 1999; Carlton et al., 2001a, 2003). Modulation of primary afferent fibers is regarded as an important therapeutic target because sensitization of peripheral nociceptors defines the quality and duration of primary hyperalgesia during inflammation (Ji et al., 2006). To investigate the possibility that cross-excitation between peripheral afferent terminals could contribute to a peripheral mechanism of secondary hyperalgesia, we previously developed a model to observe potential changes in mechanical and electrophysiological receptive properties of adjacent afferent terminals following antidromic electrical stimulation (ADES) of a cut peripheral cutaneous nerve. Using this model, we demonstrated the existence of cross-excitation between adjacent primary afferent terminals via the non-synaptic release of some excitatory neurotransmitters (Zhao et al., 1996; Zhang et al., 2001, 2008; Jia et al., 2002; Sun et al., 2002, 2003; Cao et al., 2007). The findings are consistent with reports that secondary hyperalgesia is induced by antidromic stimulation of cutaneous nerves in man and rabbit (Lewis, 1936; Fitzgerald, 1979). Lewis (1936) proposed that the peripheral mechanism for secondary hyperalgesia was the sensitization of uninjured nociceptors arising from the peripheral release and spread of pro-inflammatory mediators from stimulated nociceptors. Both excitatory and inhibitory neurotransmitters can be released from primary afferent terminals under inflammatory and nociceptive conditions (Juranek and Lembeck, 1997; Elhassan et al., 2001; Tashev et al., 2001; Tsai et al., 2002; Du et al., 2003; Olias et al., 2004; Tian et al., 2005; Zhang et al., 2006a, 2006b; Cao et al., 2007; Zhang et al., 2008). Our previous studies indicate that excitatory neurotransmitters such as glutamate and substance P (SP) are involved in the crossexcitation between primary afferent terminals. Somatostatin (SST) and its synthetic octapeptide analog octreotide have been shown to have analgesic effects through activation of SST receptors (Silveri et al., 1994; Heppelmann and Pawlak, 1997; Elhassan et al., 2001; Tashev et al., 2001; Tsai et al., 2002; Olias et al., 2004; Paran et al., 2005; Sándor et al., 2006). In addition, SST and its receptors are found in presumed nociceptive neurons of the dorsal root ganglia (Lawson, 1995) and on peripheral unmyelinated nociceptive fibers (Elhassan et al., 1998; Patel, 1999; Carlton et al., 2001a, 2003). SST can be
released from capsaicin-sensitive sensory nerve terminals (Helyes et al., 2000, 2004; Pintér et al., 2006). These studies contribute to the idea that SST and its receptors have peripheral analgesic effects. The primary purpose of the present study was to investigate whether SST modulates the efferent activity of primary afferent neurons. Because some pain models report sex differences (Berkley et al., 2006; Greenspan et al. 2007), another purpose of this study was to determine if sex differences affect the SST modulation of efferent activity.
2.
Results
2.1.
Receptive characteristics of primary afferent fibers
A total of 172 single unit recordings were obtained. Of these units, 52 were C fibers, 84 were Aδ fibers, and 36 were Aβ fibers. Mean conduction velocities (CVs) used for classification, mean mechanical receptive thresholds, and spontaneous background discharge rates are presented in Table 1. The receptive fields of all units responded to mechanical stimulation and were located 0.5 to 2.0 cm from the dorsal midline. All units exhibited very low spontaneous background discharge rates and there was no significant difference in spontaneous discharge rates among the 3 types of units (P N 0.05, one way ANOVA test followed by Dunnett's method) and there was no significant difference between male and female rats (P N 0.05, t-test).
2.2. Cross-excitation after ADES of the dorsal root from adjacent spinal segment (DRASS) In the group receiving only ADES of the DRASS (St), after stimulating the T11 dorsal root, mean mechanical thresholds of C units from the T10 and T12 spinal nerve in male and female rats decreased significantly from 0.80 ± 0.10 and 0.63 ± 0.07 mN to 0.58 ± 0.04 and 0.50 ± 0.04 mN, respectively (P b 0.01, paired t-test, shown in Fig. 1A). Mean discharge rates of C units in male and female rats significantly increased from 1.35 ± 0.15 and 1.50 ± 0.21 impulse/min to 3.08 ± 0.48 and 3.17 ± 0.35 impulse/min, respectively compared with control before the stimulation (P b 0.05, paired t-test, shown in Fig. 1B). The increased discharge rates lasted for at least 10 min following
Table 1 – Baseline characteristics of primary afferent fibers C units
Mean mechanical receptive threshold (mN) Mean conduction velocity (m/s) Spontaneous background discharge rate (impulse/min)
Aδ units
Aβ units
Male rats (n = 20)
Female rats (n = 32)
Male rats (n = 37)
Female rats (n = 47)
Male rats (n = 14)
Female rats (n = 22)
0.76 ± 0.11
0.62 ± 0.08
0.29 ± 0.05
0.26 ± 0.02
0.22 ± 0.02
0.21 ± 0.02
1.45 ± 0.07 (0.86–1.84) 1.35 ± 0.15
1.20 ± 0.07 (0.75–1.72) 1.50 ± 0.21
10.29 ± 1.77 (2.14–28.30) 1.04 ± 0.17
40.56 ± 1.83 (32.50–48.60) 1.04 ± 0.26
40.85 ± 2.41 (31.10–54.30) 0.64 ± 0.10
9.97 ± 1.00 (2.56–26.70) 0.83 ± 0.17
All values given as mean ± SEM. Values in parentheses show the range of conduction velocity.
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trical stimulation of the T11 dorsal root in male or in female rats (P N 0.05, paired t-test, Fig. 1 and 2C).
2.3.
Fig. 1 – Changes in mean mechanical threshold (A) and discharge rates (B) of C, Aδ and Aβ units following the antidromic electrical stimulation of dorsal root from adjacent spinal segment in male and female rats. M: in male rats; F: in female rats. *P < 0.05, compared with those before stimulation. (n = 20 for C units in male and n = 32 for C units in female rats, n = 37 for Aδ units in male rats, n = 47 for Aδ units in female rats, n = 14 for Aβ units in male rats and n = 22 for Aβ units in female rats).
ADES, gradually returning to the pre-stimulation level thereafter. No significant difference in spontaneous discharge rate was found between males and females (P N 0.05, t-test). Fig. 2A is an original recording showing the discharges of an activated C unit after ADES of DRASS. For Aδ units in male and female rats, the mean mechanical thresholds decreased significantly from 0.30 ± 0.03 and 0.25 ± 0.02 mN to 0.25 ± 0.02 and 0.21 ± 0.01 mN, respectively (P b 0.05, paired t-test, shown in Fig. 1A). Similar to C units, mean discharge rates significantly increased from 1.04 ± 0.17 and 0.83 ± 0.17 impulse/min to 2.51 ± 0.36 and 2.21 ± 0.30 impulse/ min after ADES (P b 0.05, paired t-test, Fig. 1B), and no significant difference was found between males and females (P N 0.05, t-test). Fig. 2B shows the discharges of an activated Aδ unit following antidromic stimulation. Mean mechanical thresholds and discharge rates of Aβ units showed no obvious change following antidromic elec-
Effects of SST on cross-excitation
In the group receiving normal saline injection followed by ADES of DRASS (Ns + St), mean mechanical thresholds of C units in male and female rats decreased from 0.68 ± 0.13 and 0.66 ± 0.09 mN to 0.51 ± 0.07 and 0.46 ± 0.05 mN (P b 0.01, paired t-test, Fig. 3A and B), similar to that in the St group. Following ADES, mean discharge rates of C units in male rats significantly increased from 1.20 ± 0.25 to 3.06 ± 0.75 impulse/min and in female rats from 0.83 ± 0.14 to 2.35 ± 0.28 impulse/min (P b 0.05, paired t-test, Fig. 4A and B), again similar to the St group. In the group receiving an injection of SST followed by ADES of DRASS (Sst + St), mean mechanical thresholds of C units in male and female rats were 0.66 ± 0.13 and 0.64 ± 0.09 mN, which were not different from the thresholds before stimulation (0.71 ± 0.07 and 0.62 ± 0.07 mN, P N 0.05, paired t-test, Fig. 3A and B). Unlike the St group, mean discharge rates following ADES were 1.84 ± 0.30 impulse/ min for C units in male rats and 1.23 ± 0.16 impulse/min for C units in female rats, not significantly different from the control discharge rates measured before ADES (1.20 ± 0.31 and 0.79 ± 0.13 impulse/min, P N 0.05, paired t-test, shown in Fig. 4A and B). In the Ns + St group following ADES, mean discharge rates of male and female Aδ units significantly increased from 0.95 ± 0.25 and 0.96 ± 0.12 impulse/min to 2.46 ± 0.63 and 2.20 ± 0.23 impulse/min, respectively (P b 0.05, paired t-test, Fig. 4C and D). In the Sst + St group, mean discharge rates of Aδ units following ADES were 1.37 ± 0.26 and 1.11 ± 0.15 impulse/min for male and female rats, respectively. No significant difference was found when compared with that before ADES (1.00 ± 0.20 and 0.65 ± 0.09, P N 0.05, paired t-test, Fig. 4C and D). After antidromic stimulation, mean mechanical thresholds of Aδ units in male and female rats significantly decreased from 0.28 ± 0.05 and 0.26 ± 0.02 mN to 0.24 ± 0.03 and 0.21 ± 0.02 mN in the Ns +St group (P b 0.05, paired t-test, Fig. 3C and D), but not in the Sst + St group (P N 0.05, paired t-test, shown in Fig. 3C and D). These results suggest that local administration of SST inhibited the cross-excitation of C and Aδ units evoked by ADES of DRASS. The inhibitory effect of SST on afferent discharge rates was not significantly different between male and female rats for C and Aδ fibers (P N 0.05, t-test). For Aβ units in the Ns + St group, mean mechanical thresholds and discharge rates did not change significantly following ADES. SST had no effect on either the mean mechanical thresholds or on the mean discharge rates of Aβ units following ADES (P N 0.05, paired t-test, Fig. 3E, F, 4E and F).
2.4. Effects of an SST receptor antagonist on cross-excitation In the group receiving co-injection of the SST receptor antagonist cyclo-somatostatin (c-SOM) prior to SST injection then followed by ADES of DRASS (c-som + Sst + St), antidromic stimulation decreased mean mechanical thresholds of
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Fig. 2 – Original recordings of discharge from a C unit (A), an Aδ unit (B) and an Aβ unit (C) following antidromic electrical stimulation of dorsal root from adjacent spinal segment. Conduction velocities of the units were 0.77, 17.7 and 43.7 m/s for C, Aδ and Aβ unit, respectively.
male and female C units from 0.60 ± 0.09 and 0.59 ± 0.12 mN to 0.41 ± 0.06 and 0.48 ± 0.12 mN (P b 0.01, pair t-test, Fig. 3A and B), and significantly increased mean discharge rates from 0.83 ± 0.38 and 1.13 ± 0.29 impulse/min to 3.83 ± 0.67 and
2.20 ± 0.61 impulse/min following ADES (P b 0.05, pair t-test, Fig. 4A and B). Mean mechanical thresholds of Aδ units in male and female rats decreased from 0.30 ± 0.06 and 0.28 ± 0.03 mN to
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Fig. 3 – Changes in mechanical threshold after antidromic electrical stimulation of the dorsal root from adjacent spinal segment in all groups. (A) C units in male rats; (B) C units in female rats; (C) Aδ units in male rats; (D) Aδ units in female rats; (E) Aβ units in male rats; (F) Aβ units in female rats.*P < 0.05, **P < 0.01, compared with those before stimulation.
0.22 ± 0.04 and 0.23 ± 0.03 mN after ADES, respectively (P b 0.01, pair t-test, Fig. 3C and D). Mean discharge rates significantly increased from 0.71 ± 0.31 and 1.13 ± 0.24 impulse/min to 3.69 ± 0.97 and 3.76 ± 0.66 impulse/min, respectively (P b 0.05, paired t-test, Fig. 4C and D). These data indicate that the SST receptor antagonist reversed the inhibitory effects of SST. In the c-som + Sst + St group, neither the mean mechanical thresholds nor the mean discharge rates of Aβ units significantly changed following ADES in male and female rats (P N 0.05, paired t-test, Fig. 3E, F, 4E and F).
2.5.
Tonic inhibitory effect of SST on the cross-excitation
In the group receiving an injection of c-SOM followed by ADES of DRASS (c-som + St), mean mechanical thresholds of C units in male and female rats significantly decreased from 0.64 ± 0.06 and 0.57 ± 0.12 mN to 0.44 ± 0.07 and 0.46 ± 0.13 mN, and mean mechanical thresholds of Aδ units significantly decreased from 0.29 ± 0.04 and 0.26 ± 0.04 mN to 0.22 ± 0.03 and 0.20 ± 0.03 mN in male and female rats following ADES (P b 0.01, paired t-test, Fig. 3A–D). Mean discharge rates in C units significantly increased from 1.50 ± 0.49 and 0.97 ± 0.22
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Fig. 4 – Changes in discharge rate after antidromic electrical stimulation of dorsal root from adjacent spinal segment in all groups. (A) C units in male rats; (B) C units in female rats; (C) Aδ units in male rats; (D) Aδ units in female rats; (E) Aβ units in male rats; (F) Aβ units in female rats. *P < 0.05, compared with those before stimulation.
impulse/min to 5.71 ± 1.60 and 4.41 ± 0.85 impulse/min in male and female rats, and mean discharge rates in Aδ units significantly increased from 1.17 ± 0.48 and 0.91 ± 0.20 impulse/min to 3.87 ± 1.14 and 4.40 ± 0.88 impulse/min in male and female rats following stimulation (P b 0.01, paired t-test, Fig. 4A–D). The mean changes in discharge rates (percentage of net change from control) for C and Aδ units
following ADES in the c-som + St group were significantly greater than that in the St and Ns + St groups (P b 0.05, one way ANOVA test followed by Dunnett's method, Fig. 5A). The mean changes in mechanical thresholds (percentage of net change from control) for C and Aδ units following ADES in the c-som + St group were significantly different from that in the St and Ns + St groups (P b 0.05, one way ANOVA test followed
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by Dunnett's method, Fig. 5B). The results suggested that SST might have been released tonically in peripheral tissues and exerted inhibitory effects on peripheral afferent terminals. For Aβ units, SST had no effect on either the mean mechanical thresholds or on the mean discharge rates in male and female rats following ADES in the c-som + St group (P N 0.05, paired t-test, Fig. 3E, F, 4E and F).
3.
Discussion
The most important finding from the present study is that local injection of SST into the receptive field of the recorded afferent nerve blocked the increase in discharge rate and the decrease in mechanical threshold of C and Aδ fibers that resulted from ADES of DRASS. In our previous studies, we demonstrated that primary afferent terminals could be activated by antidromic stimulation of an adjacent peripheral cutaneous nerve (Zhao et al., 1996; Zhang et al., 2001, 2008; Jia et al., 2002; Sun et al., 2002, 2003; Cao et al., 2007). The experimental model used in the present study had character-
Fig. 5 – The differences in mean changes in discharge rates and mechanical thresholds (percentage of net change from control) between the Ns + St and the c-som + St groups, and between the St and the c-som + St groups. (A) The mean changes of discharge rates in three types of fibers; (B) The mean changes percentage in mechanical thresholds in three types of fibers. *, P < 0.05.
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istics similar to that used in our previous studies for studying cross-excitation between primary afferent terminals in vivo. The only difference is that in the present study the dorsal root was stimulated, eliminating the possibility that cross-excitation was caused by activation of sympathetic efferents since many studies have reported the existence of sympatheticsomatosensory interactions or coupling in the peripheral nervous system (Chung et al., 1997; Baron, 2000; Xie et al., 2007). As described in previous studies (Lembeck, 1983; Holzer, 1988; Du et al., 2003), when a primary afferent fiber is excited by inflammation, injury or antidromic stimulation, some neurotransmitters and neuromodulators can be released from terminals in branches of the same fiber by the axon reflex. It seems conceivable that some neurotransmitters could also diffuse and bind to receptors on terminals of neighboring afferent fibers. Our previous studies indicate that the mean discharge rate of primary afferents significantly increases after antidromic electrical stimulation of an adjacent spinal nerve and that local injection of NMDA, nonNMDA or NK-1 receptor antagonists into the receptive field of the recorded units blocks the increase of discharge rate (Cao et al., 2007; Zhang et al., 2008). Antidromically-activated release of neurotransmitters/neuromodulators may be a neural basis for the peripheral sensitization of pain, especially for secondary hyperalgesia. It has been widely accepted that some neurotransmitters/ neuromodulators are released from unmyelinated and thinly myelinated nerve fibers following antidromic stimulation. These neurotransmitters/neuromodulators include proinflammatory mediators such as glutamate (Juranek and Lembeck, 1997; Omote et al., 1998; deGroot et al., 2000), SP, calcitonin gene-related peptide (Lembeck, 1983; Holzer, 1988; Sakaguchi et al., 1991; Juranek and Lembeck, 1997; McGillis and Fernandez, 1999), as well as anti-inflammatory mediators such as opioids (Cabot et al., 1997; Bigliardi-Qi et al., 2004) and SST (Szolcsányi et al., 1998; Carlton et al., 2001a, 2003; Helyes et al., 2004). The balance between releases of these pro- and antiinflammatory mediators may contribute to the resolution of local inflammation and hyperalgesia in peripheral tissues. Intraplantar injection of SST or the SST analogue octreotide reduces both nociceptive behaviors induced by carrageenan and formalin and responses to thermal stimulation of Cmechanoheat sensitive fibers in a dose-dependent fashion (Corsi et al., 1997; Carlton et al., 2001a, 2001b, 2003, 2004). SST also reduces responses of joint mechanoreceptors to noxious rotation of the cat knee joint and to both innocuous and noxious rotation of the inflamed knee joint (Heppelmann and Pawlak, 1997; Pawlak and Schmidt, 2004). In the present study, the effects of SST were blocked by the SST receptor antagonist c-SOM, suggesting receptor specificity in the process. In addition, ADES in the presence of c-SOM produced a greater increase in discharge rate and a greater decrease in mechanical threshold compared with ADES and saline vehicle strongly suggesting that SST was tonically released producing a cross-excitatory inhibition at peripheral nerve terminals. The results are consistent with previous studies demonstrating that SST has analgesic effects in rodents and humans by acting on peripheral SST receptors (Karalis et al., 1994; Silveri et al., 1994; Helyes
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et al., 2000, 2004; Corsi et al., 1997; Olias et al. 2004; Szolcsányi et al., 2004; Paran et al., 2005; Ji et al., 2006). SST receptors have been identified on afferent terminals and in the dorsal root ganglion (Elhassan et al., 1998; Patel, 1999; Carlton et al., 2001a, 2003). A deficiency in the sst2 subtype of SST receptor is associated with exaggerated jejunal afferent sensitivity to both mechanical and chemical stimulation in mice (Rong et al., 2007). SST, after peripheral release from nerve terminals, may affect not only other peripherally released neuropeptides but cells from other physiological systems as well. SST and the pro-inflammatory neuropeptide SP co-exist in the same neuron (Kashiba et al., 1996) and separate in different populations of primary afferent neurons (Hökfelt et al., 1976). Under inflammatory conditions, SST may inhibit the release of pro-inflammatory neuropeptides from sensory terminals and thereby attenuate nociceptive responses (Green et al., 1992; Hathway et al., 2001; Carlton et al., 2001a, 2001b; Sawynok, 2003; Weckbecker et al., 2003; Grilli et al., 2004). SST also acts on receptors of vascular endothelial and immune cells and may form communication networks between the nervous and immune systems affecting both inflammation and nociception (Krantic, 2000; Dasgupta, 2004; Pintér et al., 2006). Some studies indicate females have lower thresholds to noxious stimuli, a greater ability to discriminate stimuli, higher pain ratings, and less tolerance for noxious stimuli compared to males (Berkley et al., 2006; Greenspan et al. 2007). Injection of glutamate into the masseter muscle induces a muscle pain response which is significantly greater in females than in males (Cairns et al., 2001, 2003). Subcutaneous administration of glutamate in human subjects evokes sex-related differences in some pain responses (Gazerani et al., 2006). On the other hand, a number of studies indicate the absence of sex differences in some pain responses (Hogeweg et al., 1996; Banik et al., 2006). In the present study no obvious sex differences were present in the cross-excitation of peripheral afferent terminals following ADES of the dorsal root from the adjacent segment. In summary, the present study suggests that crossexcitation between peripheral afferent terminals exists in both male and female rats and that somatostatin has a receptor-mediated tonic inhibitory effect on the crossexcitation. Local analgesic effects of somatostatin produced by local peripheral delivery may avoid deleterious side effects in the central nervous system and provide a basis for the use of somatostatin or its analogs in the pharmacotherapy of acute and chronic painful inflammatory diseases of peripheral origin.
4.
Experimental procedures
4.1.
Animals
Experiments were performed on 97 Sprague–Dawley rats weighing 220–300 g. The animals were housed on a 12–12 h light-dark cycle, and given food and water available ad libitum. Experimental procedures were approved by the
Institutional Animal Ethics Committee of Xi'an Jiaotong University.
4.2.
Surgical procedures
Rats were anesthetized initially with urethane (1.0 g/kg i.p.); supplemental doses (0.05 g/kg/h) were given as needed to maintain areflexia. Hair on the back was removed by shaving. In order to avoid damaging receptive fields on the back, a left paramedian skin incision 1 cm away from the median line was made longitudinally to expose the right dorsal cutaneous branches of the T10 and T12 spinal nerves. The cutaneous branches were dissected and freed for 2.5–3.5 cm from the surrounding tissues. A T10-12 laminectomy was performed in order to expose and cut the right T11 dorsal root for antidromic electrical stimulation (ADES) (see Fig. 6). A pool was formed by raising the skin flaps and filling the space with warm paraffin oil (37 °C). Rectal temperature was maintained at approximately 37 ± 0.5 °C using a servo-controlled heating blanket. The isolated T10 and T12 cutaneous nerve branches were cut close to their passage through the latissimus dorsi muscle in order to block the afferent and efferent information to and from the spinal cord.
4.3.
Stimulation and recording
The distal cut end of the T11 dorsal root was placed across silver bipolar electrodes for ADES. On a small platform, the distal cut portion of the dorsal cutaneous branches of the T10 or T12 spinal nerves were mechanically desheathed and teased apart under a dissecting microscope. Small filaments were repeatedly split with sharpened watchmaker forceps until single unit activity was isolated. Neural activity from T10 or T12 was recorded using platinum bipolar electrodes, then amplified (Biophysical Amplifier AVB-11A), filtered (30– 3000 Hz), and displayed on an oscilloscope (VC-11, Nihon Kohden, Japan) for monitoring the action potential's waveform and amplitude. The signals were also fed into a computer based data acquisition system (Spike2, Cambridge Electronic Design Limited, Cambridge, UK) that allowed continuous monitoring of discharges as well as off-line data analysis. Fig. 6 summarizes the experimental approach and demonstrates the cross-excitation induced by ADES of DRASS. For all groups discharge rates prior to any intervention were recorded for 2 min and served as control values. Then the T11 dorsal root was electrically stimulated continuously for 30 s (intensity 0.6 mA, duration 0.5 ms, frequency 20 Hz). In previous references and our preliminary experiments we found this intensity excited most Aδ and some C fibers (Leem et al., 1993; Zhao et al., 1996; Kanda et al., 2001) Following ADES, discharge rates were recorded for an additional 10 min. The mechanical threshold of each unit was measured with calibrated von Frey's filaments applied to its receptive field (RF) and expressed as the minimum force (mN) needed to evoke a response in ≥50% of the trials (Cain et al., 2001; Tian et al., 2005; Zhang et al., 2006a, 2006b). The location of each unit's receptive field that was most sensitive to mechanical stimulation was marked by a felt tip pen and
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vehicle (10 μl) was slowly injected over a 5-min period after recording background discharge.
4.5.
Fig. 6 – Schematic drawing showing the experimental method and the presumed pathway for cross-excitation between peripheral nerve terminals. When afferent nerves are excited by inflammation, injury or antidromic stimulation, excitatory and inhibitory neurotransmitters can be released from peripheral terminals of the afferent nerves. Neurotransmitters released from peripheral terminals may diffuse to adjacent afferent terminals and influence the activity of adjacent terminals.
was the target for drug injection. Mechanical thresholds were determined before and after ADES. The range of von Frey filaments used in this study exerted bending forces from 0.1 mN to 42.3 mN. Primary afferents were classified by their CV. A single electrical stimulus (square wave) with progressively increasing intensity (0.1–1 mA, duration 0.5 ms) was applied to the receptive field using silver bipolar electrodes. CV was calculated by dividing the distance from the stimulating to the recording electrode by the latency of the action potential. Units with CVs b2.0 m/s were classified as C fibers, 2.0–30.0 m/s as Aδ fibers, and 30.1–70.0 m/s as Aβ fibers (Horch et al., 1977; Tian et al., 2005; Zhang et al., 2006a, 2006b).
4.4.
Groups and pharmacological intervention
Five experimental groups were used: 1) ADES of DRASS only (St); 2) normal saline injection followed by ADES of DRASS (Ns + St); 3) SST injection followed by ADES of DRASS (Sst + St); 4) co-injection of SST receptor antagonist cyclo-somatostatin (c-SOM) prior to SST and followed by ADES of DRASS (c-som + Sst + St) and 5) injection of c-SOM followed by ADES of DRASS (c-som + St). SST (Sigma Co., St. Louis, MO, USA) was diluted with saline to 0.1 mM (Heppelmann and Pawlak, 1997). c-SOM (Sigma Co., St. Louis, MO, USA) was diluted with saline to 12.8 μM (according to preliminary experiments). In order to inject drugs or vehicle into the receptive field of a recorded unit, we attached a needle (28-gauge) to a 50 μl Hamilton syringe via PE10 tubing and inserted the needle subcutaneously into most sensitive region of the receptive field. Drug or the saline
Data analysis
Discharge rates before and after ADES were counted at one minute intervals and expressed in impulse/min. All data were presented as mean ± SEM. Statistical analysis was performed with SigmaStat 2.0 software. One way ANOVA test followed by Dunnett's method was used to compare differences in spontaneous background discharge rates among the 3 types of units and differences in discharge rates and mechanical thresholds among St, Ns + St and c-som + St groups. Paired ttests were used to compare mean mechanical thresholds before and after ADES, and to compare mean discharge rates before and after ADES. An t-test was used to compare mechanical thresholds and discharge rates between male and female rats. A P-value of b0.05 was considered statistically significant.
Acknowledgments The project was supported by the National Natural Science Foundation of China (No. 30600219, 30772705) and the Special Research Fund for the Doctoral Program of High Education (No. 20070698101). REFERENCES
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