Elsevier

Brain Research

Volume 889, Issues 1–2, 19 January 2001, Pages 118-130
Brain Research

Research report
Effects of baclofen on colon inflammation-induced Fos, CGRP and SP expression in spinal cord and brainstem

https://doi.org/10.1016/S0006-8993(00)03124-3Get rights and content

Abstract

The present study demonstrates sites of expression for Fos protein in the brainstem and lumbosacral spinal cord of rats subjected to mustard oil irritation of the colon. The protective effect of baclofen, a selective GABAB receptor agonist, on the induced Fos protein increases was determined. Mustard oil injected into the lumen of the colon produces an acute site-specific inflammation. Immunocytochemical localization of Fos protein in neuronal nuclei was evident after 1 h, was greatest at 2 h and was still evident but declining at 8 h. In the spinal cord the majority of Fos labeled neurons were localized in the superficial laminae of lumbar (L6) cord with more found in the sacral (S1) cord. Some labeled neurons were also found in the deeper spinal laminae, intermediolateral nucleus and around lamina X. Brainstem sites expressing Fos included the nucleus of the solitary tract in the medulla, parabrachial, locus coeruleus, pontine and caudal dorsal raphe nuclei and periaqueductal gray. Weak Fos protein labeling existed in a few cells in vehicle control animals. Systemic administration of the GABAB receptor agonist, baclofen (10 mg/kg, i.p.), significantly reduced Fos expression in the spinal cord after mustard oil treatment but significantly increased the relative number of nuclei labeled in the nucleus of the solitary tract. Baclofen also significantly decreases dorsal horn CGRP immunoreactivity relative to the increased levels seen after inflammation of the colon. The SP content increases observed after inflammation of the colon were not altered by baclofen. These data suggest that: (1) neurons in regions important for nociceptive transmission, descending inhibitory control and autonomic control are activated by noxious stimulation of the colon, and (2) baclofen specifically reduces Fos expression in the superficial dorsal horn of the spinal cord induced by nociceptive afferent input.

Introduction

The evoked expression of the immediate–early-gene-encoded protein, Fos, one of the inducible transcription factors, serves as a quantifiable marker to identify neuronal populations activated by noxious somatic and visceral stimulation. Numerous studies of sensory function have used Fos as a marker for neurons activated by a particular noxious stimulus. In the past decade, most investigators have focused on immunocytochemical localization of Fos expression induced by noxious somatic stimulation including mechanical, thermal and chemical insult to peripheral nerve, skin or joint [19], [22], [35], [41], [42]. However, visceral pain is the primary cause of suffering and dysfunction in patients with malignant and inflammatory states. The mechanisms and neural systems involved in visceral pain have also been investigated utilizing different visceral models. Noxious distention of hollow viscera (i.e. esophagus, stomach, duodenum and colorectal) induces a specific pattern of Fos expression in rat spinal cord and hindbrain that reflects the intensity of the stimulation [28], [35], [44], [64], [66]. Other investigators have detected Fos in the midbrain and pons [23], [52] following visceral stimulation (peritoneum or heart or cardiopulmonary). The expression of Fos has also been reported in the spinal cord after noxious mechanical or chemical noxious stimulation of the urinary bladder [24], [39], lower urinary tract [10], [12] and with renal pain induced by ureteral obstruction [4]. Fos is observed at hindbrain [14] and mesodiencephalic [15] levels in cyclophosphamide cystitis as a model of visceral pain. Almeida and Lima [3] have combined retrograde labeling from the dorsal reticular nucleus with immunocytochemical detection of Fos expression in the spinal cord following chemical stimulation of the bladder to define a destination for this noxious input.

Localization of Fos expression subsequent to c-fos gene activation can be a useful tool to examine the effectiveness of different analgesic drugs after nociceptive stimulation. Recently, the anti-nociceptive effects of some drugs have been studied in animal models of persistent visceral pain using Fos expression as an independent measure. Numerous studies have demonstrated that opiate receptor agonists, especially morphine, attenuate pain related behavior and Fos expression in rat spinal cord neurons following all types of noxious stimulation [1], [22], [32], [61], including visceral pain. In some visceral pain models, such as noxious colorectal distention [65] and intraperitoneal chemical injection [34], morphine reduced Fos expression in spinal neurons and in cells of the nucleus of solitary tract as well as reduced nociceptive behavior. The effect of opioids was dose-dependent and naloxone reversible. Clonidine (α2-adrenoceptor agonist, i.v.) depressed bladder reflexes and produced some decrease in spinal Fos expression after chemical irritation of the lower urinary tract [13]. This suggests that noradrenergic pathways which are known to modulate cutaneous nociceptive perception are also involved in modulating visceral nociceptive mechanisms. The glutamatergic receptor is also very important for the induction and maintenance of spinal nociceptive events that lead to hyperalgesia following tissue damage in animal models. There is evidence that glutamatergic receptor antagonists (MK-801 and LY-215490) can reduce Fos expression induced in spinal neurons after chemical irritation of the lower urinary tract of the rat [11], [37].

In the present study Fos expression will be used to examine involvement of the gabaergic system in visceral nociception. GABA is an important inhibitory amino acid neurotransmitter in the central nervous system and plays an important role in decreasing nociceptive information at the level of the spinal cord through GABAB receptor actions. According to a review by Hammond [33], anatomical, neurochemical, and electrophysiological studies of the actions of GABA in the spinal cord assume that GABA receptors and their agonists modulate the afferent transmission of nociceptive information and thus produce antinociception. Baclofen, a selective GABAB receptor agonist, is largely used clinically to reduce spasticity but can provide some relief of pain [47]. Baclofen has been shown to reduce visceral pain arising from urinary bladder irritation induced by the instillation of xylene [2]. Sawynok and Dickson’s work [56] demonstrates that noradrenaline and 5-hydroxytryptamine receptor antagonists can reverse the antinociceptive action of baclofen in tail-flick and hot-plate tests in rats, suggesting that the antinociceptive effects of baclofen are mediated indirectly through activation of noradrenergic descending pathways to the spinal cord.

The effect of baclofen pre-treatment on spinal Fos expression invoked by persistent nociceptive stimulation has been investigated in some pain models. Systemic administration of baclofen (i.v.) reduced the number of Fos immunoreactive neurons evoked by intraplantar injection of carrageenan [18] and chemical irritation of the lower urinary tract [13]. Together the pharmacological studies provide insight into the circuitry of pain mechanisms in the dorsal horn.

The objectives of the present study are to: (1) identify regions of the neuraxis expressing the Fos protein in response to mustard oil inflammation of the distal colon, (2) assess the time-course of Fos expression and (3) compare the effect of baclofen on the spinal and medullary Fos expression induced in the same animal model.

Section snippets

Animals

All experiments were approved by Animal Care and Use Committee at our institution and are consistent with the guidelines of this committee and policies on the Ethical Treatment of Research Animals published by the International Association for the Study of Pain. Sixty-six male Spraque–Dawley rats (200–300 g) were used for the study. The animals were housed in a room with a constant ambient temperature of 22°C and a 12 h light/dark cycle with free access to food and water.

Induction of the colon inflammation

Thirty-eight rats were

Histopathological changes in the inflamed colon

The presence of inflammatory cells was minimal in the colon of the control rats treated with peanut oil. More inflammatory cells infiltrated the colon after mustard oil injection into the colon. Eosinophils and neutrophilis were found in the colon 1 h after mustard oil injection. The appearance of polymorphs, eosinophils and nuclear dust (necrotic mucosal cells) in histological sections from the colon was also evident at the 2 and 4 h time points (Fig. 1). There were only a few eosinophils in

Fos localization after colon inflammation

After induction of colon inflammation with mustard oil, Fos protein expression becomes evident as nuclear staining in cells of the lumbosacral spinal cord and the brainstem, while little stainable protein exists in vehicle control animals. Fos expression is most evident 2 h after injection of mustard oil into the colon in most of the regions where it is observed. Fos labeled cells are observed in the superficial dorsal horn of the lumbosacral spinal cord and in other regions of the brainstem

Conclusion

The results of this study provide evidence to show that when the colon is inflamed with mustard oil (1) inflammatory cells infiltrate the colon and (2) Fos protein expression is observed in regions receiving afferent input from the colon. This includes the dorsal horn of the lumbosacral spinal cord and the nucleus of the solitary tract. Brainstem regions activated as indicated by the presence of Fos labeled cells included the parabrachial complex, locus coeruleus, raphe pontis, caudal dorsal

Acknowledgements

This study was supported by Program Project Grant NS11255 and RO1 NS32778.

References (70)

  • J.G Cui et al.

    effects of spinal cord stimulation on touch-evoked allodynia involve GABAergic mechanisms. An experimental study in the mononeuropathic rat

    Pain

    (1996)
  • J.G Cui et al.

    Spinal cord stimulation attenuates augmented dorsal horn release of excitatory amino acid in mononeuropathy via a GABAergic mechanism

    Pain

    (1997)
  • J.G Cui et al.

    Effect of spinal cord stimulation on tactile hypersensitivity in mononeuropathic rats is potentiated by simultaneous GABAB and adenosine receptor activation

    Neurosci. Lett.

    (1998)
  • J.A DeLeo et al.

    Differential c-fos-like protein expression in mechanically versus chemically induced visceral nociception

    Brain Res. Mol. Brain Res.

    (1991)
  • S.J Enna et al.

    Regulation of neurokinin-1 receptor expression by GABA(B) receptor agonists

    Life Sci.

    (1998)
  • K.R Gogas et al.

    Inhibition of noxious stimulus-evoked pain behaviors and neuronal fos-like immunoreactivity in the spinal cord of the rat by supraspinal morphine

    Pain

    (1996)
  • T Herdegen et al.

    Inducible and constitutive transcription factors in the mammalian nervous system: control of gen expression by Jun. Fos and Krox, and CREB/ATF proteins

    Brain Res. Brain Res. Rev.

    (1998)
  • C.H Hubscher et al.

    Responses of neurons in caudal solitary nucleus of female rats to stimulation of vagina, cervix, uterine horn and colon

    Brain Res.

    (1994)
  • M Lanteri-Minet et al.

    Spinal and hindbrain structures involved in visceroception and visceronociception as revealed by the expression of Fos, Jun and Krox-24 proteins

    Neuroscience

    (1993)
  • Q.P Ma et al.

    Basal and touch-evoked fos-like immunoreactivity during experimental inflammation in the rat

    Pain

    (1996)
  • S.T Meller et al.

    The possible role of glia in nociceptive processing and hyperalgesia in the spinal cord of the rat

    Neuropharmacology

    (1994)
  • A Pertovaara et al.

    Induction and suppression of immediate–early genes in the rat brain by a selective alpha-2-adrenoceptor agonist and antagonist following noxious peripheral stimulation

    Neuroscience

    (1993)
  • L Rodella et al.

    Expression of Fos immunoreactivity in the rat supraspinal regions following noxious visceral stimulation

    Brain Res. Bull.

    (1998)
  • P Santicioli et al.

    Adenosine inhibits action potential-dependent release of calcitonin gene-related peptide- and substance P-like immunoreactivities from primary afferents in rat spinal cord

    Neurosci. Lett.

    (1992)
  • J Sawynok

    GABAergic mechanisms in antinociception

    Pro. Neuropsychopharmacol. Biol. Psychiatry

    (1984)
  • J Sawynok et al.

    Evidence for the involvement of descending noradrenergic pathways in the antinociceptive effect of baclofen

    Brain Res.

    (1985)
  • M Shafizadeh et al.

    Involvement of GABAB receptors in the antinociception induced by baclofen in the formalin test

    Gen. Pharmacol.

    (1997)
  • A.F Sved et al.

    Endogenous GABA acts on GABAB receptors in nucleus tractus solitarius to increase blood pressure

    Brain Res.

    (1990)
  • T.R Tolle et al.

    Opiates modify induction of c-fos proto-oncogene in the spinal cord of the rat following noxious stimulation

    Neurosci. Lett.

    (1990)
  • R.J Traub et al.

    Fos-like proteins in the lumbosacral spinal cord following noxious and non-noxious colorectal distention in the rat

    Pain

    (1992)
  • R.J Traub et al.

    Differential expression of c-fos and c-jun in two regions of the rat spinal cord following noxious colorectal distention

    Neurosci. Lett.

    (1993)
  • R.J Traub et al.

    Noxious distention of viscera results in differential c-Fos expression in second order sensory neurons receiving ‘sympathetic’ or ‘parasympathetic’ input

    Neurosci. Lett.

    (1994)
  • R.J Traub et al.

    Attenuation of c-fos expression in the rat lumbosacral spinal cord by morphine or tramadol following noxious colorectal distention

    Brain Res.

    (1995)
  • R.J Traub et al.

    Noxious colorectal distention induced-c-Fos protein in limbic brain structures in the rat

    Neurosci. Lett.

    (1996)
  • R.J Traub et al.

    Differential c-fos expression in the nucleus of the solitary tract and spinal cord following noxious gastric distention in the rat

    Neuroscience

    (1996)
  • Cited by (55)

    • Abdominal Pain

      2022, Comprehensive Pharmacology
    • Differential responses of neurons in the rat caudal ventrolateral medulla to visceral and somatic noxious stimuli and their alterations in colitis

      2019, Brain Research Bulletin
      Citation Excerpt :

      The increased convergent inflow to the CVLM, we revealed, may at least partly be a consequence of such functional plasticity in the spinal cord, but we cannot ruled out that in colitis unknown yet neuroplastic changes facilitating viscerosomatic convergence may occur also at the caudal medullary level, for example, at synapses of spinal afferents or local excitatory interneurons that modulate nociceptive inflow to CVLM cells. We cannot exclude also that the enhanced visceral and somatic nociceptive transmission through the CVLM can be a result of increased excitatory inputs to the region from the nucleus of the solitary tract (Aicher et al., 1995; Nosjean et al., 2002) or from other pain-regulating brainstem centers, such as the locus coeruleus, dorsal raphe nucleus, and periaqueductal gray (Cobos et al., 2003; Ossipov et al., 2010; Tavares and Lima, 2007), the heightened activity of which has been demonstrated in the presence of colonic inflammation (Jain et al., 2015; Lu and Westlund, 2001; Lyubashina et al., 2018). We believe that the colitis-induced changes in response properties of CVLM nociceptive neurons described above may lead to the pathological mixing of medullary visceral and somatic nociceptive processing that, apart from promoting referred somatic hyperalgesia in colitis, can be concerned with the generalization of autonomic responses to different types of pain.

    • Colitis-induced alterations in response properties of visceral nociceptive neurons in the rat caudal medulla oblongata and their modulation by 5-HT3 receptor blockade

      2018, Brain Research Bulletin
      Citation Excerpt :

      We have found that colonic inflammation evoked a substantial increase in the percentage of CRD-excited neurons and their noxious stimulation-evoked responses in both the CVLM and NTS. These observations are in accordance with previous findings, demonstrating elevated c-Fos expression in the NTS in colitis (Lu and Westlund, 2001). It is noteworthy that in our experiments in inflamed animals no significant differences in the magnitude of excitatory responses to CRD were detected between CVLM and NTS neurons, indicating their equal reactivity to abdominal pain signals.

    • Altered intrinsic and synaptic properties of lumbosacral dorsal horn neurons in a mouse model of colitis

      2017, Neuroscience
      Citation Excerpt :

      For example, experimental rodent models of colonic inflammation can induce visceral and somatic hyperalgesia (Laird et al., 2001b; Lamb et al., 2006). Inflammation also increases neural activation (cFos and pERK), and the release of ‘pain’ neuropeptides (Substance P and CGRP) within the spinal cord dorsal horn (Lu and Westlund, 2001; Sun and Luo, 2004; Traub et al., 2008; Harrington et al., 2012). In addition, functional studies using in vivo extracellular recording have observed that following inflammation, dorsal horn neurons exhibit increased spontaneous action potential (AP) discharge (Al-Chaer et al., 2000; Ness and Gebhart, 2001; Qin et al., 2005) and increased AP discharge in response to distension of the colon (Laird et al., 2001a; Wang et al., 2005b).

    • The 5-HT<inf>4</inf> receptor-mediated inhibition of visceral nociceptive neurons in the rat caudal ventrolateral medulla

      2017, Neuroscience
      Citation Excerpt :

      The cholinergic signaling, in turn, is shown to mediate the bidirectional modulation of cortical and subcortical GABA release exerted by BIMU8 and other 5-HT4 receptor agonists (Bianchi et al., 2002; Cai et al., 2002). By employing these mechanisms, the drug might enhance cortical antinociceptive outflow (Ohara et al., 2005; Cao et al., 2008; Jurik et al., 2015) and facilitate descending cholinergic and GABAergic inhibition of visceral nociception (Kawamata et al., 1993; Hara et al., 1999; Thor et al., 2000; Lu and Westlund, 2001; Sengupta et al., 2002; Brusberg et al., 2009), promoting thereby a decline in the excitability of visceral nociceptive neurons in the caudal medulla. Meanwhile, it has been shown that the BIMU8-induced reduction of number of abdominal constrictions in mice was not prevented by pretreatment with reserpine, a monoamine store depletory (Ghelardini et al., 1996).

    View all citing articles on Scopus
    View full text