Preclinical Studies

16 May

The analysis of pain mechanisms in preclinical animal models of inflammatory and neuropathic pain has shown that iGluRs and mGluRs play important roles in peripheral  sensitization,  central sensitization,  and descending pain mod- ulation (Fisher et al. 2000; Fundytus 2001; Carlton 2001; Willis 2001; Parsons

2001; Neugebauer and Carlton 2002; Neugebauer 2002; Varney and Gereau

2002; Ruscheweyh and Sandkuhler 2002; Lesage 2004; Woolf 2004).

Ionotropic Glutamate Receptors


Peripheral administration of NMDA or non-NMDA receptor antagonists  in- hibits nociceptive behavior in the second phase of the formalin test and thermal and mechanical hyperalgesia associated with inflammation  of the hindpaw (complete  Freund’s adjuvant  CFA- or carrageenan-induced) and knee joint (kaolin/carrageenan-induced) (Carlton  2001; Parsons 2001; Du et al. 2003). NMDA receptor antagonists tested in these studies include the clinically used compounds dextrorphan (a metabolite of dextromethorphan), ketamine, and memantine.  Peripheral  NMDA receptors  might also be involved in visceral pain, as memantine was shown to inhibit the responses of single fibers in the decentralized pelvic nerves to colorectal distention (McRoberts et al. 2001). In a model of neuropathic  pain, block of peripheral  NMDA receptors (by MK-

801), but not non-NMDA receptors  (by NBQX), attenuated  and delayed the onset of mechanical hyperalgesia (Jang et al. 2004). The potential therapeutic value of peripheral NMDA, AMPA, and kainate receptor antagonists still awaits a systematic analysis in models of inflammatory and, particularly, neuropathic pain.

Spinal Cord

Spinal administration of NMDA or non-NMDA antagonists is well known to inhibit  central sensitization  in various models of inflammatory  and neuro- pathic pain (Fisher et al. 2000; Fundytus  2001; Willis 2001; Parsons  2001). NMDA or non-NMDA receptor  antagonists  in the spinal cord reversed the enhanced  responses  of dorsal horn  neurons,  including  spinothalamic  tract cells, in inflammatory  pain states induced  by intradermal  capsaicin, intra- plantar  carrageenan,  or intraarticular kaolin  and  carrageenan  (Stanfa and Dickenson 1999; Fisher et al. 2000; Fundytus 2001; Willis 2001; Parsons 2001; Schaible et al. 2002). Compounds  tested in these studies  include the clini- cally used NMDA receptor antagonists ketamine and memantine, a somewhat AMPA receptor-preferring antagonist (NBQX), and a GluR5 kainate receptor- selective antagonist (LY382884). Central sensitization of dorsal horn neurons, including spinothalamic  tract cells, in models of neuropathic  pain has also been shown to be inhibited by spinal administration of NMDA (memantine, dextrorphan, and MK-801) and non-NMDA (CNQX and LY382884) receptor antagonists (Carlton et al. 1997; Carlton et al. 1998; Parsons 2001; Palecek et al.

2004; Willis and Coggeshall 2004).

Behavioral data also show that NMDA and non-NMDA receptor antagonists are antinociceptive in inflammatory and neuropathic pain models (Fisher et al.

2000; Fundytus 2001; Willis 2001; Parsons 2001; Soliman et al. 2005). Spinally or systemically administered  NMDA receptor antagonists, including memantine and dextromethorphan, inhibit  nociceptive behavior, thermal  hyperalgesia, and mechanical allodynia associated with peripheral inflammation  as well as visceral hypersensitivity. NMDA receptor antagonists also reduce heat hyper- algesia and, less consistently, mechanical allodynia associated with peripheral nerve injury. Interestingly,  intrathecal  administration of an NR2B subunit- selective antagonist (CP-101606) inhibited mechanical allodynia in a model of postoperative  pain (Nishimura  et al. 2004) but had no effect on mechanical allodynia in a neuropathic  pain model whereas memantine  was antinocicep- tive in that model (Nakazato et al. 2005), suggesting differential roles of spinal NR2B in different forms of pain. Systemic administration of another  agent with NR2B-selective antagonistic properties  (ifenprodil) had antinociceptive effects in inflammatory and neuropathic pain models but, unlike MK-801 and memantine, did not inhibit NMDA-evoked responses of dorsal horn neurons (Nakazato et al. 2005). Thus the role of spinal NR2B subunit-containing NMDA receptors in nociceptive processing remains unclear.

Intrathecally or systemically administered non-NMDA receptor antagonists, including NBQX, have antinociceptive effects in animal models of inflamma- tory and neuropathic  pain, but they also produce side effects such as ataxia.

A selective AMPA receptor antagonist (GYKI53655), however, did not produce antinociception in the formalin pain test at doses that did not cause ataxia (Sim- mons et al. 1998), suggesting that kainate rather than AMPA receptors may be

useful targets. Indeed, more recent studies focused on kainate receptors and showed that systemic administration of a GluR5/6-selective antagonist (SYM

2081; 2S,4R-4-methylglutamic acid; Sutton et al. 1999) and GluR5-selective

antagonists  such as LY382884 (Simmons et al. 1998) and orally active com- pounds LY467711 and LY525327 (Dominguez et al. 2005) inhibited nociceptive behavior in the formalin test, thermal hyperalgesia, and mechanical allodynia induced by carrageenan or capsaicin, and thermal hyperalgesia and mechani- cal allodynia in a model of peripheral nerve injury. The site or sites of action of these systemically administered drugs remain to be determined.


In the brainstem  (RVM), NMDA receptor activation is involved in descend- ing facilitation whereas inhibition involves activation of non-NMDA receptors (Ren and Dubner 2002; Gebhart 2004; Vanegas and Schaible 2004). NMDA- dependent descending facilitation appears to be particularly important for the development of secondary thermal hyperalgesia and mechanical allodynia in the early stages of inflammatory  pain; it may be important for maintenance rather  than  initiation  of neuropathic  pain  states as well (Ren and  Dubner

2002; Porreca et al. 2002; Heinricher et al. 2003; Gebhart 2004; Vanegas and

Schaible 2004). Microinjection of NMDA receptor antagonists such as AP5 into the RVM inhibited hyperalgesic behavior produced  by injection of formalin into the hindpaw, carrageenan-induced hindpaw inflammation, application of mustard oil to the hindlimb skin (topical), and colon inflammation. In contrast, block of non-NMDA receptors in the RVM further enhanced the hyperalgesic response, suggesting the presence of descending inhibition. Differential effects of NMDA and non-NMDA receptor antagonists on antinociceptive “off-cells” and  pronociceptive  “on-cells” in the RVM also suggest that  distinct  phar- macological profiles of inhibitory  and facilitatory circuits may be a neural mechanism of bidirectional descending control (Heinricher  et al. 2003). De- scending modulation  may undergo time-dependent functional changes such that facilitation decreases or inhibition increases (or both) at the later stages of inflammatory pain. These changes and their role in primary versus secondary hyperalgesia await a detailed analysis.

The role of iGluRs in the PAG in prolonged or chronic pain states is less well understood. Block of NMDA receptors in the PAG inhibited pain in the formalin

test, but not the hotplate test, suggesting that NMDA receptors are involved in tonic, but not phasic, pain (Vaccarino et al. 1997). In another study, however, NMDA receptor antagonists in the PAG were ineffective in the formalin test but activation of NMDA receptors produced antinociception  (Berrino et al. 2001).

The role of non-NMDA receptors in the PAG remains to be determined.

In higher brain areas, intrathalamic injection of an NMDA receptor antago- nist (AP5) reduced thermal and mechanical hyperalgesia in the acute and sub- acute phases of the carrageenan-induced hindpaw inflammation  model. The effects were confined to the withdrawal responses evoked from the injected paw. Pretreatment with intrathalamic injections of antisense oligodeoxynucleotides directed against the NR1 subunit prevented the development of thermal hyper- algesia and attenuated the development of mechanical hyperalgesia (Kolhekar et al. 1997). The pharmacology  of thalamic pain mechanisms  has yet to be analyzed at the single cell level in preclinical pain models.

In the amygdala both NMDA (AP5) and non-NMDA (NBQX) receptor antag- onists inhibited the nociceptive responses of CeLC neurons in the kaolin/carra- geenan-induced arthritis pain model (Li and Neugebauer 2004b). However, in- creased function of NMDA receptors rather than non-NMDA receptors appears to be a key mechanism  of pain-related  synaptic plasticity recorded in brain slices from arthritic animals compared to naïve controls (Bird et al. 2005).

In the anterior cingulate cortex (ACC), bilateral microinjections of an NMDA receptor antagonist (AP5) significantly inhibited bilateral mechanical allody- nia in the CFA-induced inflammatory pain model (posttreatment on day 1; Wei et al. 2002). Conversely, overexpression of NR2B in the forebrain, including the ACC and insular cortex, increased formalin-induced  pain behavior and me- chanical allodynia associated with CFA-induced hindpaw inflammation  (Wei et al. 2001). However, pretreatment with bilateral injections of NMDA (AP5) or non-NMDA (DNQX) receptor  antagonists  into the ACC had no effect on

acute nociceptive behaviors in the formalin test whereas affect-related behav- ior (formalin-induced conditioned place avoidance) was effectively eliminated by intra-ACC microinjection of AP5 but not DNQX (Lei et al. 2004).

Metabotropic Glutamate Receptors


There is good evidence to suggest the involvement and endogenous activation of peripheral  group I mGluRs in models of inflammatory  and neuropathic pain but not in normal  nociception  (Carlton  2001; Neugebauer 2001, 2002; Neugebauer and Carlton 2002; Varney and Gereau 2002; Lesage 2004). Periph- eral injections of antagonists  for mGluR1 (including  CPCCOEt) or mGluR5 (MPEP) inhibited nociceptive behavior in the second, but not the first, phase of the formalin test. Similarly, peripheral  administration of MPEP reversed mechanical allodynia in carrageenan-  and CFA-induced inflammatory  pain models. The antagonists  did not inhibit normal nociception  and had no ef- fect when injected into the contralateral non-inflamed paw. Peripheral group I mGluRs may also play a role in neuropathic  pain. In the spinal nerve ligation model, peripheral injections of an mGluR5 antagonist (SIB-1757, 6-methyl-2- (phenylazo)-3-pyridinol) into the injured hindlimb, but not the contralateral paw, reversed thermal hyperalgesia, but not mechanical allodynia, and had no effect in sham-operated animals.

Peripheral  injection  of a group  II mGluR agonist (APDC) inhibited  no- ciceptive behavior  in  the  first  and  the  second  phase  of formalin-induced pain (Neugebauer  and Carlton 2002). APDC also blocked prostaglandin  E2 (PGE2)-induced thermal  hyperalgesia and PGE2- and carrageenan-induced mechanical allodynia (Yang and Gereau 2002; Yang and Gereau 2003). How- ever, APDC had no effect on basal thermal and mechanical thresholds in naïve animals. The antinociceptive  effects of APDC were antagonized  by a selec- tive group  II antagonist  (LY341495) when tested. Importantly,  blockade of peripheral  group II mGluRs (LY341495) prolonged PGE2- and carrageenan- induced  mechanical allodynia, suggesting that peripheral  group II mGluRs mediate  endogenous  anti-allodynic  effects (Yang and  Gereau 2003). Simi-

larly, peripheral  injection  of a group  II/III  antagonist  (MSOPPE, (RS)-α- methylserine-O-phosphate monophenyl  ester) enhanced  glutamate-induced

mechanical allodynia (Neugebauer and Carlton 2002). In the spinal nerve le- sion model of neuropathic  pain, pre-treatment with APDC delayed the on- set of mechanical  allodynia  but  post-treatment with APDC had  no  effect (Jang et al. 2004). The contribution of peripheral  mGluRs to  nociceptive

processes  associated  with inflammatory  and  neuropathic  pain  would sug- gest that peripherally acting group I antagonists  and group II agonists may

have a therapeutic  value in the  treatment  of pain  states  arising  from  pe- ripheral  tissue injury.  The role of group  III mGluRs remains  to be deter- mined.

Spinal Cord

The endogenous activation and involvement of spinal group I mGluRs, par- ticularly mGluR1, in prolonged nociception and persistent pain states is well documented in behavioral and electrophysiological studies using antagonists, antibodies, and antisense oligonucleotides in models of inflammatory pain (in- duced by formalin, intradermal  capsaicin, intraplantar carrageenan  or CFA, and  intraarticular kaolin/carrageenan) and  neuropathic  pain  (Neugebauer et al. 1999; Fundytus 2001; Karim et al. 2001; Neugebauer 2001; Neugebauer

2002; Varney and Gereau 2002; Lesage 2004; Soliman et al. 2005). Electro- physiological studies of spinal dorsal horn neurons, including spinothalamic

tract  cells, showed antinociceptive  effects of spinally administered  group  I

antagonists, including mGluR1-selective compounds (CPCCOEt), or antisense oligodeoxynucleotides directed against mGluR1, in pain-related  central sen- sitization following intradermal  capsaicin, topical application of mustard  oil to the skin, or intraarticular kaolin/carrageenan injections (Neugebauer et al.

1999; Fundytus 2001; Neugebauer 2001; Varney and Gereau 2002). The involve- ment and intrinsic activation of mGluR5 in spinal neurons in pain models is

not entirely clear yet.

Intrathecal  administration of group  I antagonists,  including  CPCCOEt- (mGluR1) and MPEP (mGluR5), inhibited nociceptive behavior in the forma- lin test, thermal hyperalgesia and mechanical allodynia following intraplantar carrageenan, and capsaicin-induced mechanical, but not thermal, hypersensi- tivity (Fundytus 2001; Neugebauer 2001; Neugebauer 2002; Varney and Gereau

2002; Lesage 2004; Soliman et al. 2005). Group I antagonists had also antinoci- ceptive effects in models of neuropathic  pain. Whereas mGluR1-selective an- tagonists inhibited  thermal hyperalgesia and mechanical allodynia, the role

of mGluR5 in neuropathic  pain is controversial, but a number of studies have shown antinociceptive effects of systemically or intrathecally applied blockers of mGluR5 (Varney and Gereau 2002; Lesage 2004). When directly compared,

systemic administration of an mGluR1 antagonist  [LY456236, (4-methoxy- phenyl)-(6-methoxy-quinazolin-4-yl)-amine HCl] reversed mechanical allo- dynia in a neuropathic  pain model whereas mGluR5 antagonists  (including

MPEP) only attenuated  it; however, both strongly inhibited formalin-induced nociceptive behavior (Varty et al. 2005). It is possible that mGluR5 receptors play a more prominent  role in inflammatory than neuropathic pain states, but mGluR1 appears to be important for both.

The roles of group II and group III mGluRs in spinal pain mechanisms are less clear. Intrathecal  administration of a group III agonist (LAP4) produced

antinociceptive  effects in the second phase of the formalin test whereas in- trathecally administered group II agonists such as LY379268 were ineffective in this pain model (Neugebauer and Carlton 2002; Varney and Gereau 2002; Jones et al. 2005). Intrathecal administration of group II (APDC) and III (LAP4) ago- nists inhibited mechanical allodynia following intradermal  capsaicin but had little effect on thermal hyperalgesia (Soliman et al. 2005). Electrophysiological studies showed that activation of spinal group II mGluRs inhibited electrically evoked nociceptive responses of dorsal horn neurons in carrageenan-induced hindpaw inflammation whereas mixed effects (inhibition and facilitation) were observed in control  rats. A selective group II agonist (LY379268) inhibited capsaicin-induced central sensitization of spinothalamic tract cells but had no effect on the responses of non-sensitized neurons (Neugebauer et al. 2000). In contrast, a group III agonist (LAP4) inhibited the responses of spinothalamic tract cells to brief innocuous and noxious stimuli under normal condition as well as capsaicin-induced central sensitization (Neugebauer et al. 2000). These data suggest a dramatic change in the functional role of spinal group II, rather than group III, mGluRs in inflammatory pain.


In the brainstem,  administration of group I (DHPG) and II (LCCG) agonists into the PAG decreased the nociceptive response in the second phase of the formalin test. These antinociceptive  effects were antagonized  by the respec- tive antagonists  (CPCCOEt, group I mGluR1; EGLU, group II) (Maione et al.

2000). A group III agonist (LSOP) increased the formalin-evoked nociceptive response. This facilitatory effect was antagonized  by a group III antagonist (MSOP). Interestingly, an antagonist  for mGluR5 (MPEP) potentiated  per se the early nociceptive phase of the formalin test, suggesting mGluR5 in the PAG is endogenously  activated to produce  descending inhibition  (Berrino et al.

2001). EGLU and MSOP had no effects on their own. It has been suggested that group I and group II mGluRs in the PAG positively modulate descending pain inhibition whereas group III mGluRs inhibit this antinociceptive pathway (Maione et al. 2000; Berrino et al. 2001).

In higher brain areas, the function of mGluRs in prolonged or chronic pain states is still largely unknown. Recent electrophysiological and behavioral stud-

ies in the amygdala suggested an important role for group I mGluRs, partic- ularly mGluR1, in nociceptive plasticity and pain behavior (Neugebauer et al.

2004). In the kaolin/carrageenan arthritis  pain model, administration of an- tagonists selective for mGluR1 (CPCCOEt) or mGluR5 (MPEP) into the amyg- dala inhibited  the increased  responses  of sensitized CeLC neurons  whereas the responses of these neurons under normal conditions before arthritis were

inhibited by MPEP but not by CPCCOEt (Li and Neugebauer 2004a). Accord- ingly, CPCCOEt had no effect on basal synaptic transmission in CeLC neurons

recorded  in slices from normal  animals but inhibited  pain-related  synaptic plasticity in slices from arthritic rats. MPEP inhibited basal synaptic transmis- sion as well as synaptic plasticity (Neugebauer et al. 2003). Thus, enhanced receptor activation of mGluR1 appears to be a key mechanism of pain-related synaptic plasticity in the CeLC. Importantly, behavioral studies that addressed the significance of mGluR1 and mGluR5 function in the amygdala showed that CPCCOEt administration into the CeLC inhibited higher integrated behavior organized in the amygdala (vocalization afterdischarges) and in the brainstem (vocalizations during  stimulation)  as well as spinal nociceptive withdrawal reflexes. MPEP inhibited only vocalizations organized in the amygdala. It ap- pears that activation of mGluR1 in the amygdala contributes to pain produc- tion through descending facilitation whereas mGluR5 is involved in intrinsic amygdala processes (Han and Neugebauer 2005). The roles of group II and III mGluRs in nociception and plasticity in the amygdala remain to be determined.

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