The primary finding in the current study was that spinal delivery of the highly selective 5-HT3 receptor antagonist ondansetron between14 and 30 days following L5/6 spinal nerve ligation in the rat failed to reduce the thermal or mechanical hypersensitivity that accompanies this neuropathic pain state. Secondly, we used immunohistochemical and biochemical approaches to assess alterations in the spinal serotonergic system. We didn’t observe alterations in spinal serotonin content, density of serotonergic terminals, or distribution of 5-HT3 receptors following SNL. These results were unanticipated and surprising considering the recent interest in 5-HT3 receptor antagonists as potential analgesics (Greenshaw and Silverstone, 1997
; McCleane et al., 2003
; Suzuki et al., 2004b
; Thompson and Lummis, 2007
) and several preclinical studies that demonstrate a facilitatory role for spinal 5-HT3 activation in the setting of tissue injury (Svensson et al., 2006
) and central neuropathic pain states (Chen et al., 2009
; Oatway et al., 2004
; Suzuki et al., 2004a
). Failure of intrathecal ondansetron to alter hypersensitivity across three behavioral endpoints and two laboratories led us to abandon neurotoxicity screening of this compound for intrathecal study in humans under NIH grant GM48085.
It is well known that descending pathways that originate in the rostral ventral medulla (RVM) are essential for maintaining mechanical hypersensitivity and spinal neuronal excitability that accompanies a variety of persistent pain states including neuropathic pain (Porreca et al., 2002
; Suzuki et al., 2002
; Vera-Portocarrero et al., 2006
). A component of descending facilitation following peripheral nerve injury is likely due to enhanced serotonergic drive originating in the RVM and subsequently activating spinal 5-HT3 receptors (Marinelli et al., 2002
; Suzuki et al., 2004a
). The most compelling evidence for this comes from in vivo
electrophysiological studies. Suzuki and colleagues reported that pharmacological blockade of 5-HT3 receptors with spinal ondansetron strongly inhibits mechanical and to a lesser degree thermal evoked neuronal responses of WDR neurons in deep laminae (V-VI) of rats 14 days following SNL compared to sham operated rats. This enhanced 5-HT3 receptor activation relies on a spinobulbar circuit involving neurokinin 1 (NK1) receptor bearing neurons within the superficial aspects of the spinal cord (Suzuki et al., 2004a
) as ablation of spinal NK1 neurons abolishes the inhibitory effects of ondansetron. Similar physiological evidence for enhanced serotonergic facilitation of spinal excitability has been observed following injection of formalin in the hindpaw (Suzuki et al., 2002
), bone cancer (Donovan-Rodriguez et al., 2006
), and osteoarthritis (Rahman et al., 2009
A facilitatory role of spinal 5-HT3 receptor activation on pain related behaviors has also been described in several persistent pain states (Zeitz et al., 2002
). Depletion of serotonergic nerve fibers reduces the delayed (phase II) nocifensive pain behaviors that occur following injection of formalin into the hindpaw (Svensson et al., 2006
) as well as thermal and mechanical hypersensitivity associated with central (Oatway et al., 2004
) and peripheral neuropathic pain states (Rahman et al., 2006
). Transgenic knockout and pharmacological studies using 5-HT3 receptor subtype selective antagonists have begun to address the 5-HT receptor subtypes responsible for these behavioral facilitatory effects. Spinal ondansetron in rats (Svennson et al., 2006
; Suzuki 2002
) or genetic deletion of 5-HT3 receptors in mice (Zeitz 2005) reduced phase II formalin responses. Additionally, administration of ondansetron in the formalin model reduced spinal levels of phosphorylated ERK providing evidence that 5-HT3 receptor activation contributes to sensitization of spinal cord neurons (Svensson et al., 2006
). Spinal ondansetron administered acutely (Oatway et al., 2004
) or chronically (Chen et al., 2009
) has also been shown to effectively reduce mechanical allodynia in rats with spinal cord injury as well as thermal and mechanical allodynia that develops in rats following chronic opioid administration (Vera-Portocarrero et al., 2007
Despite these positive finding studies, preclinical studies examining the effects of ondansetron on symptoms of neuropathic pain have produced conflicting results. Recently, Dogrul and colleagues reported that acute spinal ondansetron (10 μg) produced a sustained attenuation of thermal and mechanical hypersensitivity following L5/6 SNL in rats. In contrast to these findings, Okazaki and colleagues reported that spinal administration of the 5-HT3 receptor antagonist CGP35348 had no effect on established mechanical allodynia in rats (Okazaki et al., 2008
). Furthermore, mice deficient in 5-HT3 receptors develop a similar degree of mechanical allodynia as wild type mice following partial sciatic nerve injury and peripheral inflammation (Zeitz et al., 2002
). These studies suggest that spinal facilitatory behavioral effects are not universally observed in all persistent pain states or consistently in neuropathic models of peripheral nerve injury. In the current study, we addressed this controversy by examining more than one 5-HT3 receptor selective antagonist in a battery of behavioral tests. We observed that spinal ondansetron (1, 3, 10, 30, and 100 μg) and dolasetron (40 μg) did not reverse thermal or mechanical hypersensitivity when administered acutely between 14-30 days following SNL.
Several explanations for the differences between our findings and those reported by Dogrul and colleagues are possible. Both studies examined ondansetron using a similar dose, drug formulation, and method of administration. In the study by Dogrul spinal ondansetron was administered at 7 days post SNL while in our study we initiated ondansetron between 14 -30 days following SNL. It is possible that 5-HT3 mediated facilitatory effects are time dependent and resolve between 14 and 30 days post SNL. However, physiological studies demonstrate that enhanced serotonergic drive is still present 14 days following SNL (Suzuki et al., 2004a
). Secondly, Dogrul et al. do not comment whether behavioral assays were conducted in a blinded manner. In the current study, all behavioral assays were conducted by an investigator blind to treatment and negative results were replicated using blinded conditions in a second laboratory. In both laboratories, positive controls were included which showed clear efficacy. Unintended experimenter bias has been suggested to have an impact on behavioral outcomes in preclinical studies. Notably, negative and/or positive outcomes of pharmacological studies examining the efficacy of spinal opioids in preclinical models of neuropathic pain closely paralleled the consensus on the efficacy of spinal opioids reported simultaneously in clinical studies (Eisenach and Lindner, 2004
). It is also possible that subtle differences in surgical procedures may impact behavioral outcomes between labs. There is considerable variability in the extent of nerve injury and onset of mechanical hypersensitivity in the L5/6 SNL model (Chung et al., 2004
). In the study by Dogrul the baseline levels of mechanical hypersensitivity as measured using von Frey filaments are slightly lower than baseline values in the current study (1.6 ± 0.7 gm vs. 4.1 ± 0.7 gm). Thus, the degree of mechanical hypersensitivity may be important for recruiting descending serotonergic drive sufficient to observe an inhibitory effect of ondansetron. Likewise, the degree of nerve injury may have a pronounced impact on spinal plasticity of the serotonergic system.
Multiple factors may impact the net effect of 5-HT3 receptor activation in the spinal cord including plasticity of serotonergic terminals, alterations in 5HT3 receptor distribution, as well as the nature and intensity of peripheral stimuli (Tyce and Yaksh, 1981). Following dorsal rhizotomy sprouting of serotonergic terminals has been observed in segmental regions of the spinal cord corresponding to deafferented terminals but not in regions with intact afferents (Polistina et al., 1990
; Ramer et al., 2007
). Following chronic constriction injury to the sciatic nerve bilateral increases in serotonin content were observed between seven and 14 days postoperatively (Satoh and Omote, 1996
) coinciding with the development of mechanical hypersensitivity in this model. Following injury to the spinal cord an increase in the density of serotonergic terminals is observed rostral to the lesion site which has been causally linked to mechanical hypersensitivity in corresponding dermatomes (Oatway et al., 2004
). In a recent study, spinal levels of 5-HT and its metabolite transiently decreased in the dorsal ipsilateral lumbar spinal cord of L5/6 SNL rats seven days postoperatively but recovered to baseline levels by 28 days. These alterations coincided with decreased inhibitory effects of 5-HT on C fiber evoked responses of WDR neurons (Liu et al., 2010
). It is unclear why both increases and decreases in descending serotonergic system have been described following nerve injury, however this may be due to differences in magnitude or type of peripheral nerve injury as spinal cord injury, dorsal rhizotomy at multiple spinal segments and chronic constriction of the sciatic nerve involve injury to a larger percentage of afferents innervating the spinal cord compared to L5/6 SNL.
In our study, we did not observe alterations in serotonin content or density of serotonergic terminals consistent with the negative behavioral effects of ondansetron (14 days post SNL). We also did not observe alterations in the density of 5-HT3 receptors using immunohistochemical approaches. One limitation of our approach is that we do not discriminate between alterations of 5-HT3 receptor on terminals of primary afferent fibers or populations of interneurons. Although a recent study in model of osteoarthritis provides evidence that 5-HT3 receptor mRNA was unchanged in the dorsal root ganglia of rats with osteoarthritis despite enhanced physiological effects of ondansetron in this model (Rahman et al., 2009
) suggesting alterations in 5-HT3 receptor on primary afferents are not required for enhanced serotonergic drive.
Our behavioral results also disagree with physiological results that demonstrate facilitatory effects of 5-HT3 receptor activation on mechanically evoked neuronal responses of deep dorsal horn WDR neurons in SNL animals (Suzuki et al., 2004a
). When comparing physiological results to behavioral outcome measures it is important to consider the differences between these outcome measures. Notably, behavioral measures of mechanical hypersensitivity assess threshold responses whereas physiological studies of mechanically evoked neuronal responses involve application of suprathreshold stimuli. Suprathreshold or more intense stimuli may be necessary to recruit descending serotonergic facilitatory drive required to see an inhibitory effect of ondansetron on neuronal activity. Thus, behavioral assessment of withdrawal thresholds employed in the current study may not be sensitive enough to detect facilitatory effects of 5-HT3 receptors in the spinal cord.
Another possibility for the lack of behavioral effect compared to physiologic studies may be due to dual effects of 5-HT3 receptor activation on distinct population of neurons within the spinal cord dorsal horn resulting in a net lack of effect on behavioral outcomes. Previous electrophysiological studies assessing the effects of ondansetron on neuronal responses following SNL focused on WDR neuron responses in deeper laminae (V-VI). It should be noted that deep WDR neurons only comprise a subpopulation of ascending pathways that contribute to the behavioral manifestation of mechanical hypersensitivity (Keller et al., 2007
; Ossipov et al., 2000
; Sun et al., 2001
). Several recent functional studies demonstrate that the dominant effect of 5-HT3 receptor activation in the spinal cord is anti-nociceptive by promoting GABAergic inhibitory synaptic transmission (Alhaider et al., 1991
; Giordano, 1991
; Giordano and Schultea, 2004
). Specifically, it has been reported in rats that activation of 5-HT3 receptors in the spinal cord mediates the release of GABA but not glycine or glutamate (Kawamata et al., 2003
). Recently, Fukushima and colleagues using transgenic GAD67-GFP reporter mice and in vitro
spinal cord slice physiology demonstrated that 5-HT3 receptor activation induces outward currents in GABAergic interneurons and increases evoked and spontaneous inhibitory postsynaptic currents in a GABAA
receptor dependent manner. lnterestingly, intrathecal administration of the selective 5-HT3 receptor agonist chlorophenylbiquanide (m-CPBQ) in rats with L5 SNL reverses established mechanical allodynia in a GABA A
dependent manner (Okazaki et al., 2008
) suggesting that spinal 5-HT3 receptor activation in the context of a neuropathic pain state still possesses inhibitory effects on nociceptive processing. It is not clear if similarly endogenous spinal 5-HT3 receptor activation has analgesic effects in the setting of nerve injury. In the current study this appears not to be the case as spinal ondansetron did not further reduce thermal and mechanical withdrawal thresholds following SNL. However, reduced withdrawal latency to noxious thermal stimuli has been observed in naïve mice following spinal administration of ondansetron (Scott et al., 2006
). Peripheral nerve injury results in decreased inhibitory GABAergic tone in neurons within the superficial spinal cord in part due transient disruption of chloride homeostasis (Miletic and Miletic, 2008
). Downregulation of the potassium chloride cotransporter KCC2 in superficial postsynaptic neurons has been shown to shift GABAA
mediated signaling from hyperpolarizing to depolarizing leading to increased neuronal excitability (Coull et al., 2003
; Price et al., 2005
). This may in part explain enhanced facilitation at the physiological level previously observed in SNL rats; however it is unclear how this would impact behavioral outcomes at the systems level. Future studies are warranted to examine the effects of endogenous 5-HT3 receptor activation on GABAergic nociceptive transmission and on distinct populations of dorsal horn neurons in both normal and SNL rats.
In the current study, descending serotonergic inputs acting via 5-HT3 receptor did not contribute to thermal or mechanical hypersensitivity following SNL. Examination of the spinal cord revealed no significant changes in the density of serotonergic nerve fibers or serotonin content in the dorsal horn of SNL rats compared to naïve rats. Additionally, the 5-HT3 receptor levels in the spinal cord dorsal horn were not different between SNL and naïve rats. These results agree with some more limited behavioral studies and suggest that spinal blockade of 5-HT3 receptor is insufficient to inhibit behavioral measures of mechanical hypersensitivity following experimental SNL.